Techniques for predicting, detecting and reducing aspecific protein interference in assays involving immunoglobulin single variable domains

ABSTRACT

This invention provides, and in certain specific but non-limiting aspects relates to: assays that can be used to predict whether a given ISV will be subject to protein interference as described herein and/or give rise to an (aspecific) signal in such an assay (such as for example in an ADA immunoassay). Such predictive assays could for example be used to test whether a given ISV could have a tendency to give rise to such protein interference and/or such a signal; to select ISV&#39;s that are not or less prone to such protein interference or to giving such a signal; as an assay or test that can be used to test whether certain modification(s) to an ISV will (fully or partially) reduce its tendency to give rise to such interference or such a signal; and/or as an assay or test that can be used to guide modification or improvement of an ISV so as to reduce its tendency to give rise to such protein interference or signal; —methods for modifying and/or improving ISV&#39;s to as to remove or reduce their tendency to give rise to such protein interference or such a signal; —modifications that can be introduced into an ISV that remove or reduce its tendency to give rise to such protein interference or such a signal; ISV&#39;s that have been specifically selected (for example, using the assay(s) described herein) to have no or low(er)/reduced tendency to give rise to such protein interference or such a signal; modified and/or improved ISV&#39;s that have no or a low(er)/reduced tendency to give rise to such protein interference or such a signal.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/128,681, filed Mar. 4, 2014, which is a national stage filing under35 U.S.C. § 371 of international application PCT/EP2012/062251, filedJun. 25, 2012, which was published under PCT Article 21(2) in English,which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisionalapplication No. 61/500,360, filed Jun. 23, 2011, U.S. provisionalapplication No. 61/500,464, filed Jun. 23, 2011, and U.S. provisionalapplication No. 61/541,368, filed Sep. 30, 2011; PCT/EP2012/062251 alsoclaims the benefit under 35 U.S.C. § 120 of international applicationPCT/EP2011/067132, filed Sep. 30, 2011, U.S. application Ser. No.13/435,567, filed Mar. 30, 2012 and now issued as U.S. Pat. No.8,703,135, and international application PCT/EP2012/061304, filed Jun.14, 2012. The disclosures of all of the foregoing applications areincorporated by reference herein in their entireties.

The present invention relates to the field of immunoglobulin singlevariable domains.

An immunoglobulin single variable domain or “ISV” is generally definedherein as an amino acid sequence that:

-   -   comprises an immunoglobulin fold or that, under suitable        conditions (such as physiological conditions) is capable of        forming an immunoglobulin fold (i.e., by folding), i.e., so as        to form an immunoglobulin variable domain (such as, for example,        a VH, VL or VHH domain);        and that    -   forms (or under such suitable conditions is capable of forming)        an immunoglobulin variable domain that comprises a functional        antigen binding site (in the sense that it does not require an        interaction with another immunoglobulin variable domain (such as        a VH-VL interaction) to form a functional antigen binding site).

Some examples of immunoglobulin single variable domains that arecurrently known in the art are VHH's and/or (other) Nanobodies, dAb'sand (single) domain antibodies. Of these, as of the date of filing ofthe present application, various Nanobodies are in phase I and phase IIclinical trials. This makes it important to have available reliableassays for analyzing biological samples from people that are treatedwith ISV's (such as clinical trial subjects and, after such ISV's reachthe market, patients that are treated with such ISV's).

This is not only important for regulatory purposes, but also for thetreatment of patients with biological drugs, because the clinicians thatprescribe the treatment would also like to have available reliableassays to monitor various aspects of the treatment.

For example, in the clinical development of biological drug molecules,it is important to assess their immunogenic potential, and in particularthe degree to which they can elicit so-called “anti-drug antibodies” or“ADA's”. This is determined using so-called “anti-drug antibody” or “ADA(immuno)assays” (see for example the review by Shankar et al., Journalof Pharmaceutical and Biomedical Analysis, 48 (2008), 1267-1281; as wellas Mire-Sluis et al., J. Immunol. Meth. 289 (2004), 1-16; Peng et al.,Journal of Pharmaceutical and Biomedical Analysis, 54, (2011), 629-635;and Loyet et al., J. Immunol. Meth. 345 (2009), 17-28. Such ADA assaysand methods for performing them are standard knowledge in the field ofpharmacology and they are routinely used during the clinical developmentof biological drug products (as well as being required by variousregulatory agencies around the world).

For example, as described on pages 3 and 4 of the article by Mire-Sluisand as for example also exemplified schematically in the Figures of thearticle by Peng, a number of different ADA assay formats are known, suchas “ELISA-bridging format”, “ELISA-Direct Format”, “Indirect Format”,Radio Immuno-precipitation Assay (RIP), “Surface Plasmon Resonance” and“Electrochemiluminescence-Bridging Format”. Other formats for performingADA immunoassays will be clear to the skilled person.

The skilled person will also be familiar with a number of differentcommercially available technology platforms that have been shown to besuitable for setting up and performing ADA assays. These include but arenot limited to the MSD platform (Mesoscale), Gyrolab (Gyros) and theoctet platform (Fortebio).

Some non-limiting examples of ADA assay formats are also schematicallyshown in FIGS. 1A to 1C.

Generally, it should be noted that in such ADA assays for detecting ormeasuring ADA's against an ISV, the ISV is used as the “analyticalagent” (i.e., as the compound used to detect whether any ADA's arepresent in the sample that is tested), and the ADA's are the “antigen”(i.e., the compound to be detected in the sample that is tested). Thus,in these assays, the ISV will usually/often be bound to the carrier(such as the ELISA plate), whereas the ADA's (if any) will be present inthe sample that is subjected to the assay.

To better understand the invention described herein, it should alreadybe noted that—by contrast—in the methods that are used herein to predictwhether an ISV will give rise to protein interference, the ISV willusually be used as the “antigen” (i.e., as the compound to be detected),and an antibody (which is as further described herein) is used as the“analytical agent” (i.e., as a means to detect whether a given ISV bindsor not, respectively; and thus has a high or increased risk of givingrise to protein interference or not, respectively). Thus, in this methodaccording to the invention, the antibody used as analytical agent (whichis also referred to herein as the “analytical antibody”) will usually bebound to the carrier (i.e., to the ELISA plate) and the ISV will be(present in) the sample to be tested. However, it should generally benoted that the invention is not limited to assays in which the“analytical antibody” is bound to the carrier. For example, in analternative way of performing an assay according to the invention (Asshown for instance in FIG. 1 and described in the Examples), theanalytical antibody is instead used as a bridging agent and thus will bein solution rather than bound to the plate (although it is indirectlybound to the plate via the ISV that is coated on the plate). However,also in the specific bridging assay described in the Examples (which isa competitive assay) the analytical antibody is still used as theanalytical agent (i.e., to determine whether the ISV of interest bindsor not, respectively; and thus has a high or increased risk of givingrise to protein interference or not, respectively). It is also envisagedthat, based on the further disclosure herein, the skilled person will beable to design other assay formats in which the analytical antibody canbe used as an analytical agent in order to determine whether a given ISVcan bind or not, respectively; and thus has a high or increased risk ofgiving rise to protein interference or not).

As a result of research into single chain Fv's or “ScFv's” (which areconstructs that contain immunoglobulin single variable domains that,similar to ISV's, are not associated with constant domains), it has beendescribed in the art that the C-terminus of an immunoglobulin variabledomain forms a hydrophobic patch that in an antibody is buried in theinterface between the variable domain and the constant domain but thatbecomes solvent-exposed when the variable domain is not associated witha constant domain (Nieba et al., Protein Engineering, 10, 435-444(1997)). It has also been described that the exposed C-terminus may formB-cell epitopes which can give rise to and/or interact with (emergingand/or pre-existing) anti-drug antibodies (WO 11/07586), the presence ofwhich can then be determined using the ADA assays referred to above. Forthis reason, it has been proposed to make mutations to some of the aminoacid residues that form part of the C-terminus of the variable domainsto reduce said hydrophobicity and/or to remove said epitopes. Forexample, Nieba et al. suggest to mutate positions 11, 14, 41, 84, 87and/or 89 of a VH region (numbering according to Kabat), whereas in WO11/07586 it is suggested to mutate positions 99, 101 and/or 148 (AHonumbering) of a VL domain or positions 12, 97, 98, 99, 103 and/or 144 ofa VH domain (again AHo numbering—these positions correspond to positions11, 83, 84, 85, 89 and 103 according to Kabat).

However, neither of these references recognizes that certain proteinspresent in the blood or serum of a subject can interfere with ADA assaysinvolving ISV's, and because of this these references are not directedto (nor offer a solution for) the problem of how to avoid aspecificprotein interference in such ADA assays so as to allow the ADA assay tobe used to determine the true presence/extent of (arising orpre-existing) anti-drug antibodies in the sample to be tested.

By contrast, the present invention provides methods and assays thateasily allow the skilled person to predict whether an immunoglobulinsingle variable domain will or will not have a tendency to undergoaspecific protein interference in an ADA assay. The methods and assaysdescribed herein also allow the skilled person, when it is found that avariable domain may have a tendency or risk to undergo such proteininterference in an ADA assay, to easily test (proposed) modifications toa variable domain in order to predict whether any such (proposed)modifications will reduce or essentially completely avoid such proteininterference.

The present invention also describes a number of modifications that canbe made to variable domains in order to reduce or essentially avoid suchprotein interference. According to one non-limiting aspect, thismodification involves adding a limited number (as further describedherein) of amino acid residues (as further described herein) to theC-terminal end of the variable domain. Surprisingly, it has been foundthat, for a number of different variable domains or constructs basedthereon, even adding a single amino acid residue to the C-terminal end(such as a single alanine residue) can substantially or even essentiallycompletely remove the problem of protein interference in ADA assays,even though adding one such amino acid is by itself is not sufficient to“cover” or “bury” the hydrophobic patch that according to Nieba et al.is present at the C-terminus of an ISV. Similarly, but without wishingto limit the invention in any way or to any mechanism or explanation, isalso assumed that adding one such amino acid would not be sufficient to“cover” or “bury” any B-cell epitopes that according to WO 11/07586 maybe present at the C-terminus of a variable domain It should also benoted that, although according to this specific aspect of the presentinvention, adding a limited number or even a single amino acid at theC-terminus of the variable domain (i.e. without making any substitutionswithin the C-terminal region itself, as proposed by Nieba et al and WO11/07586) may—and in many cases will—significantly reduce or evenessentially remove the problem of aspecific protein interference, it isalso within the scope of this aspect of the invention that suchadditions to the C-terminal end are combined with mutations in theC-terminal region. In this respect, however, it should also be notedthat the invention is not particularly limited as to the rationalebehind making such mutations. For example, it is well known to makemutations to amino acid residues within the C-terminus (including atthose positions that are explicitly referred to by Nieba et al. and inWO 11/07586) in order to humanize a variable domain (including, withoutlimitation, a V_(HH) domain) or in order to “camelize” a V_(H) domain(reference is for example made to WO 08/020079 and some of the otherapplications by Ablynx N.V. referred to herein).

It is envisaged that the methods, assays and modifications taught hereincan be applied to any variable domain that is not linked to or otherwiseassociated with a constant domain (or with another group or peptidemoiety that functions to “shield”, cover or “bury” the C-terminal regionof the variable domain) and more generally to any variable domain thathas a C-terminal regions that is solvent-exposed. However, according toone preferred, but non-limiting aspect of the invention, the methods,assays and modifications may in particular be applied to heavy chainvariable domains (V_(H) domains), and according to one specific aspectof the invention to V_(HH) domains.

It is also envisaged that the methods, assays and modificationsdescribed herein can be suitably applied to protein constructs thatcontain one or more variable domains, and in particular to suchconstructs in which a variable domain forms the C-terminal part of theconstruct or, in the case of the methods and assays described herein, inwhich the C-terminal region of a variable domain is otherwisesolvent-exposed. Again, according to one preferred, but non-limitingaspect of the invention, the methods, assays and modifications areapplied to constructs in which a V_(H) domain (and in particular aV_(HH) domain) forms the C-terminal part of the construct or, in case ofthe methods and assays of the invention, is otherwise solvent-exposed.

Some non-limiting examples of such constructs are multivalent,multispecific (such as bispecific) or multiparatopic (such asbiparatopic) constructs that contain two or more ISV's linked directlyor via one or more suitable linkers (with again, according to onespecific aspect, a V_(H) or V_(HH) domain) forming the C-terminal partof such a construct. For example, and without limitation, such aconstruct may be entirely comprised of V_(H) domains, and in particularof Nanobodies (i.e. V_(HH) domains, humanized V_(HH) domains orcamelized V_(H) domains), again linked directly or via one or moresuitable linkers. For some non-limiting examples of such constructs anda general teaching on how such constructs can be made (in particularbased on Nanobodies) reference is for example made to Conrath et al.,JBC 276, 10(9), 7346 (2001) as well as to the review article byMuyldermans. Reviews in Mol. Biotechnol., 74: 27 (2001).

However, it is for example also envisaged that the invention can beapplied to other constructs which have a solvent-exposed variable domainand in particular have a variable domain at their C-terminus, such asfor example single chain Fv's, and in particular ScFv's that have theirheavy chain variable domain at the C-terminus.

In the present specification and claims, terms like “ISV”, “analyticalagent” and “protein interference” have the meaning as further definedherein.

In particular, an ISV as described herein may in particular either be aNanobody or an(other) ISV (i.e. other than a Nanobody) that is a VHdomain or that comprises a VH domain; and is preferably a Nanobody.

Also, any protein or polypeptide that comprises an ISV (such as anISV-based drug) preferably has said (or at least one) such ISV at itsC-terminal end. Again, said ISV may in particular either be a Nanobodyor an(other) ISV (i.e. other than a Nanobody) that is a VH domain orthat comprises a VH domain; and is preferably a Nanobody.

The invention described herein is in particular intended and suitable tobe applied to ISVs that comprise, are based on and/or have been derivedfrom heavy chain variable domains, such as VH domains (including humanVH domains) and Nanobodies such as VHH domains (including humanized andsequence optimized VHH domains) or camelized VH domains. These may besynthetic (for example, obtained starting from a synthetic libraryand/or based on a fixed framework regions), semi-synthetic (for example,humanized, camelized or sequence-optimized, or obtained by affinitymaturation or CDR grafting, starting from a natural VH or VHH domain) orfully naturally occurring VH or VHH domains. The invention willtherefore be further described herein with reference to ISV's that are,are based on and/or have been derived from VH or VHH domains.

In establishing the present invention, it has been found that in someassays (such as, for example, in ADA immunoassays) that are used foranalyzing biological samples (such as blood samples including wholeblood, serum and plasma, ocular fluid, bronchoalveolar fluid/BALF,cerebrospinal fluid or other samples of biological fluids) proteininterference may occur, and that such protein interference may give riseto an aspecific signal in some of these assays and/or in some of thesesamples. It has also been found that such protein interference may occurnot only in samples that were obtained from subjects that have beentreated with ISV's (and in particular with Nanobodies; or with proteins,polypeptides or other biological drugs that comprise at least one suchISV or Nanobody) and/or to whom the same have been administered (such aspatients or clinical trial subjects), but also in samples from subjectsthat have never received an ISV (indicating that such interference islikely due to an aspecific protein-protein interaction with pre-existingproteins rather than any emerging ADA's).

Although it has been found that such protein interference and/or such asignal in such assays is not associated with any change or reduction inpharmacological properties (such as pharmacokinetic/PK orpharmacodynamic/PD properties) of the ISV's, it would be desirable tohave techniques available for predicting, detecting, reducing and/or ifpossible avoiding such aspecific protein interference. This is thegeneral objective of the present invention.

In particular, the invention provides, and in certain specific butnon-limiting aspects relates to:

-   -   assays that can be used to predict whether a given ISV will be        subject to such protein interference and/or give rise to such an        (aspecific) signal in such an assay (such as for example in an        ADA immunoassay). Such predictive assays could for example be        used to test whether a given ISV could have a tendency to give        rise to such protein interference and/or such a signal; to        select ISV's that are not or less prone to such protein        interference or to giving such a signal; as an assay or test        that can be used to test whether certain modification(s) to an        ISV will (fully or partially) reduce its tendency to give rise        to such interference or such a signal; and/or as an assay or        test that can be used to guide modification or improvement of an        ISV so as to reduce its tendency to give rise to such protein        interference or signal;    -   methods for modifying and/or improving ISV's to as to remove or        reduce their tendency to give rise to such protein interference        or such a signal;    -   modifications that can be introduced into an ISV that remove or        reduce its tendency to give rise to such protein interference or        such a signal;    -   ISV's that have been specifically selected (for example, using        the assay(s) described herein) to have no or low(er)/reduced        tendency to give rise to such protein interference or such a        signal;    -   modified and/or improved ISV's that have no or a low(er)/reduced        tendency to give rise to such protein interference or such a        signal.

For example, in a first non-limiting aspect, the invention relates to amethod that can be used to predict whether a given ISV or Nanobody (orISV-based or Nanobody-based drug) will give rise to (or has an high orincreased risk of giving rise to) protein interference in an immunoassay(i.e., after it has been administered to a subject, a sample of abiological fluid has been obtained from said subject, and said sample issubjected to an immunoassay as further described herein), said methodcomprising performing an immunoassay that at least comprises the stepsof:

-   -   (i) contacting said ISV or Nanobody (or ISV-based or        Nanobody-based drug) with an antibody that has been obtained        from a human subject and that has been selected, generated        and/or isolated based on its ability to recognize and/or bind to        the C-terminal end of an ISV or Nanobody (the “analytical        antibody”); and    -   (ii) determining whether said ISV or Nanobody (or ISV-based or        Nanobody-based drug) is bound by said antibody in said        immunoassay.

In this method, when the ISV, Nanobody, ISV-based drug or Nanobody-baseddrug is bound by said analytical antibody, it can be expected that saidISV, Nanobody, ISV-based drug or Nanobody-based drug will give rise to(or has a high or increased risk of giving rise to) such proteininterference (as further defined herein). Based on this, for example,said ISV, Nanobody, ISV-based drug or Nanobody-based drug may bemodified or improved so as to reduce or remove its tendency to give riseto such protein interference (which may again be determined using theassay above), and some strategies that can be used to modify said ISV,Nanobody, ISV-based drug or Nanobody-based drug will be described herein(and for example include attaching a small number of amino acid residuesto the C-terminal end and/or introducing one or more specific amino acidsubstitutions).

Thus, generally, the invention makes available to the skilled personassays and methods/techniques that can be used to predict the tendencyof an ISV, Nanobody, ISV-based drug or Nanobody-based drug to give riseto protein interference and/or as a tool to improve ISVs so as to reduceor avoid their tendency to give rise to protein interference. In doingso, the invention also provides the skilled person with means to selectISV's, Nanobodies, ISV-based drugs or Nanobody-based drugs based ontheir low or reduced ability (or the absence of any ability) to giverise to protein interference. Thus, the invention provides the skilledperson with an important assay and tool that can be used in theoptimization and development of ISV's, Nanobodies, ISV-based drugs orNanobody-based drugs.

Also, as further described herein, the invention teaches the skilledperson a number of ways in which an ISV, Nanobody, ISV-based drug orNanobody-based drug can be modified or improved so as to reduce or avoidtheir tendency to give rise to protein interference. Thus, the inventionalso generally makes available modified and/or improved ISV's,Nanobodies, ISV-based drugs or Nanobody-based drugs with a reduced, lowor without any tendency to give rise to protein interference.

As further described herein, the invention can in particular be used topredict whether a given ISV or Nanobody (or ISV-based or Nanobody-baseddrug) will give rise to protein interference (as further describedherein) in an immunoassay, and more in particular in an ADA assay. SaidADA assay may for example be an ADA assay for detecting or measuringADA's against ISV's generally, and may in particular be an ADA assay fordetecting or measuring ADA's against the ISV used in steps (i) and (ii)above.

Again, as mentioned herein, an ISV as described herein may in particulareither be a Nanobody or an(other) ISV (i.e. other than a Nanobody) thatis a VH domain or that comprises a VH domain; and is preferably aNanobody.

Also, any protein or polypeptide that comprises an ISV (such as anISV-based drug) preferably has said (or at least one) such ISV at itsC-terminal end. Again, said ISV may in particular either be a Nanobodyor an(other) ISV (i.e. other than a Nanobody) that is a VH domain orthat comprises a VH domain; and is preferably a Nanobody.

The sample that is tested in said immunoassay or ADA assay is alsoreferred to herein as the “test sample” or “assay sample”. To avoidconfusion, such as “test sample” or “assay sample” should not beconfused with the biological sample that is used herein as a startingmaterial for obtaining the (polyclonal or monoclonal) “analyticalantibody” used in the invention.

In one particular preferred but non-limiting aspect, the invention canbe used to predict whether a given ISV or Nanobody (or ISV-based orNanobody-based drug) will give rise to protein interference (as furtherdescribed herein) in an immunoassay (and in particular, in an ADA assay)that involves the use of such an ISV. Again, said ADA assay may forexample be an ADA assay for detecting or measuring ADA's against ISV'sgenerally, and may in particular be an ADA assay for detecting ormeasuring ADA's against the ISV used in steps (i) and (ii) above.

In an even more particular but non-limiting aspect, the invention can beused to predict whether a given ISV or Nanobody (or ISV-based orNanobody-based drug) will give rise to protein interference (as furtherdescribed herein) in an immunoassay (and in particular, in an ADA assay)that is intended to determine or measure whether the sample contains anyADA's against the ISV. Again, for example, such an immunoassay may beone of the known types of ADA assay (for which reference is for examplemade to the prior art on ADA assays cited herein) that is performed todetermine or measure whether any ADA's against said ISV are present inthe “test sample”, wherein said test sample is a sample of biologicalfluid (as described herein) that is obtained from a subject to whichsaid ISV has been administered (as further described herein).

As further described herein, in all these aspects (and the furtheraspects of the invention described herein), the invention can also beused to select ISV's that are not or less prone to such proteininterference in such immunoassays or ADA assays; as an assay or testthat can be used to test whether certain modification(s) to an ISV will(fully or partially) reduce its tendency to give rise to suchinterference in such immunoassays or ADA assays; and/or as an assay ortest that can be used to guide modification or improvement of an ISV soas to reduce its tendency to give rise to such protein interference insuch immunoassays or ADA assays.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

In the present specification, whenever the term “ISV” is used, it shouldbe understood that:

-   -   such an ISV is preferably a Nanobody, in which the term        “Nanobody” is generally as defined in or WO 08/020079 or WO        09/138519, and thus in a specific aspect generally denotes a        VHH, a humanized VHH or a camelized VH (such as a camelized        human VH) or generally a sequence optimized VHH (such as e.g.        optimized for chemical stability and/or solubility, maximum        overlap with known human framework regions and maximum        expression). It is noted that the terms Nanobody or Nanobodies        are registered trademarks of Ablynx N.V. and thus may also be        referred to as Nanobody® and/or Nanobodies®);    -   the term “ISV” in its broadest sense also includes “ISV-based        biologicals” and, when the ISV is a Nanobody, “Nanobody-based        biologicals”. An “ISV-based biological” is defined herein as a        protein, polypeptide or other biological drug that comprises or        essentially consist of at least one (such as one, two or three)        ISV's. Similarly, a “Nanobody-based biological” is defined as a        protein, polypeptide or other biological drug that comprises or        essentially consist of at least one (such as one, two or three)        Nanobodies. As with the term “ISV”, whenever the term “ISV-based        biological” is used, it should be understood that such an        ISV-based biological is preferably a Nanobody-based biological.        Within the context of the present invention, both an “ISV-based        biological” and a “Nanobody-based biological” may for example be        a monovalent, bivalent (or multivalent), bispecific (or        multispecific), and biparatopic (or “multiparatopic) ISV        construct or Nanobody construct, respectively. Also, any        ISV-based or Nanobody-based biological may for example, in        addition to the one or more (such as one, two or three) ISV's or        Nanobodies, optionally further comprise one or more (such as one        or two) other further therapeutic moieties and/or one or more        (such as one or two) other moieties that influence the        pharmacokinetic or pharmacodynamic properties of the ISV-based        or Nanobody-based biological (such as its half-life). Suitable        examples of such further therapeutic or other moieties will be        clear to the skilled person, and for example generally can        include any therapeutically active protein, polypeptide or other        binding domain or binding unit, as well as for example        modifications such as those described on pages 149 to 152 of WO        09/138159. An ISV-based biological or Nanobody-based biological        is preferably a therapeutic or intended for use as a therapeutic        (which includes prophylaxis and diagnosis) and for this purpose        preferably contains at least one ISV against a therapeutically        relevant target (such as for example RANK-L, vWF, IgE, RSV,        CXCR4, IL-23 or other interleukins, etc.). For some specific but        non-limiting examples of such ISV-based or Nanobody-based        biologicals, reference is for example made to the various        applications by Ablynx N.V. (such as for example and without        limitation WO 2004/062551, WO 2006/122825, WO 2008/020079 and WO        2009/068627), as well as for example (and without limitation) to        applications such as WO 06/038027, WO 06/059108, WO 07/063308,        WO 07/063311, WO 07/066016 and WO 07/085814. Also, in the        present specification, unless explicitly mentioned otherwise        herein, all terms mentioned herein have the meaning given in WO        09/138519 (or in the prior art cited in WO 09/138519) or WO        08/020079 (or in the prior art cited in WO 08/020079). Also,        where a method or technique is not specifically described        herein, it can be performed as described in WO 09/138519 (or in        the prior art cited in WO 09/138519) or WO 08/020079 (or in the        prior art cited in WO 08/020079).

In particular, the following terms have the same meaning as given on,and/or where applicable can be determined in the manner described in,pages 62-75 of WO 09/138519: “agonist”, “antagonist”, “inverse agonist”,“non-polar, uncharged amino acid residue”, “polar uncharged amino acidresidue”, “polar, charged amino acid residue”, “sequence identity”,“exactly the same” and “amino acid difference” (when referring to asequence comparison of two amino acid sequences), “(in) essentiallyisolated (form)”, “domain”, “binding domain”, “antigenic determinant”,“epitope”, “against” or “directed against” (an antigen), “specificity”and “half-life”. In addition, the terms “modulating” and “to modulate”,“interaction site”, “specific for”, “cross-block”, “cross-blocked” and“cross-blocking” and “essentially independent of the pH” are as definedon (and/or can be determined as described on) pages 74-79 of WO10/130832 of applicant. Also, when referring to a construct, compound,protein or polypeptide of the invention, terms like “monovalent”,“bivalent” (or “multivalent”), “bispecific” (or “multispecific”), and“biparatopic” (or “multiparatopic”) may have the meaning given in WO09/138.519, WO 10/130832 or WO 08/020079.

The term “half-life” as used herein relation to an ISV, Nanobody,ISV-based biological, Nanobody-based biological or any other amino acidsequence, compound or polypeptide can generally be defined as describedin paragraph o) on page 57 of WO 08/020079 and as mentioned thereinrefers to the time taken for the serum concentration of the amino acidsequence, compound or polypeptide to be reduced by 50%, in vivo, forexample due to degradation of the sequence or compound and/or clearanceor sequestration of the sequence or compound by natural mechanisms. Thein vivo half-life of an amino acid sequence, compound or polypeptide ofthe invention can be determined in any manner known per se, such as bypharmacokinetic analysis. Suitable techniques will be clear to theperson skilled in the art, and may for example generally be as describedin paragraph o) on page 57 of WO 08/020079. As also mentioned inparagraph o) on page 57 of WO 08/020079, the half-life can be expressedusing parameters such as the t½-alpha, t½-beta and the area under thecurve (AUC). In this respect it should be noted that the term“half-life” as used herein in particular refers to the t½-beta orterminal half-life (in which the t½-alpha and/or the AUC or both may bekept out of considerations). Reference is for example made to theExperimental Part below, as well as to the standard handbooks, such asKenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook forPharmacists and Peters et al, Pharmacokinete analysis: A PracticalApproach (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi& D Perron, published by Marcel Dekker, 2nd Rev. edition (1982).Similarly, the terms “increase in half-life” or “increased half-life” asalso as defined in paragraph o) on page 57 of WO 08/020079 and inparticular refer to an increase in the t½-beta, either with or withoutan increase in the t½-alpha and/or the AUC or both.

When a term is not specifically defined herein, it has its usual meaningin the art, which will be clear to the skilled person. Reference is forexample made to the standard handbooks, such as Sambrook et al,“Molecular Cloning: A Laboratory Manual” (2nd. Ed.), Vols. 1-3, ColdSpring Harbor Laboratory Press (1989); F. Ausubel et al, eds., “Currentprotocols in molecular biology”, Green Publishing and WileyInterscience, New York (1987); Lewin, “Genes II”, John Wiley & Sons, NewYork, N.Y., (1985); Old et al., “Principles of Gene Manipulation: AnIntroduction to Genetic Engineering”, 2nd edition, University ofCalifornia Press, Berkeley, Calif. (1981); Roitt et al., “Immunology”(6th. Ed.), Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt'sEssential Immunology, 10th Ed. Blackwell Publishing, U K (2001); andJaneway et al., “Immunobiology” (6th Ed.), Garland SciencePublishing/Churchill Livingstone, N.Y. (2005), as well as to the generalbackground art cited herein.

Also, herein, the amino acid residues of a Nanobody are numberedaccording to the general numbering for VH domains given by Kabat et al.(“Sequence of proteins of immunological interest”, US Public HealthServices, NIH Bethesda, Md., Publication No. 91), as applied to VHHdomains from Camelids in the article of Riechmann and Muyldermans, J.Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195; or referred toherein. According to this numbering, FR1 of a Nanobody comprises theamino acid residues at positions 1-30, CDR1 of a Nanobody comprises theamino acid residues at positions 31-35, FR2 of a Nanobody comprises theamino acids at positions 36-49, CDR2 of a Nanobody comprises the aminoacid residues at positions 50-65, FR3 of a Nanobody comprises the aminoacid residues at positions 66-94, CDR3 of a Nanobody comprises the aminoacid residues at positions 95-102, and FR4 of a Nanobody comprises theamino acid residues at positions 103-113. [In this respect, it should benoted that—as is well known in the art for VH domains and for VHHdomains—the total number of amino acid residues in each of the CDR's mayvary and may not correspond to the total number of amino acid residuesindicated by the Kabat numbering (that is, one or more positionsaccording to the Kabat numbering may not be occupied in the actualsequence, or the actual sequence may contain more amino acid residuesthan the number allowed for by the Kabat numbering). This means that,generally, the numbering according to Kabat may or may not correspond tothe actual numbering of the amino acid residues in the actual sequence.Generally, however, it can be said that, according to the numbering ofKabat and irrespective of the number of amino acid residues in theCDR's, position 1 according to the Kabat numbering corresponds to thestart of FR1 and vice versa, position 36 according to the Kabatnumbering corresponds to the start of FR2 and vice versa, position 66according to the Kabat numbering corresponds to the start of FR3 andvice versa, and position 103 according to the Kabat numberingcorresponds to the start of FR4 and vice versa.].

Alternative methods for numbering the amino acid residues of VH domains,which methods can also be applied in an analogous manner to VHH domainsfrom Camelids and to Nanobodies, are the method described by Chothia etal. (Nature 342, 877-883 (1989)), the so-called “AbM definition” and theso-called “contact definition”. However, in the present description,aspects and figures, the numbering according to Kabat as applied to VHHdomains by Riechmann and Muyldermans will be followed, unless indicatedotherwise.

It should also be noted that the Figures, any Sequence Listing and theExperimental Part/Examples are only given to further illustrate theinvention and should not be interpreted or construed as limiting thescope of the invention and/or of the appended claims in any way, unlessexplicitly indicated otherwise herein.

It should further be noted that the present invention is notspecifically limited to any causation, explanation, hypothesis ormechanism of/for the protein interference (and/or signals arising inimmunoassays) that is observed in, and that may be reduced according to,the present invention. However, it is assumed that the blood or serum(or other biological fluids, such as those mentioned herein) of certainindividuals or groups of individuals may contain certain (pre-existing)proteins that under certain circumstances may (aspecifically) bind toISV's leading to a interfering signal in certain assays that are used toanalyze blood or serum samples obtained from such individuals. This isinter alia based on the observation made in establishing the presentinvention that the aspecific protein interference that is addressed bythe present invention not only occurs when assaying samples that havebeen obtained from subjects to which an ISV has previously beenadministered, but also when assaying sample that have been obtained fromsubjects that have not previously received an ISV.

In particular, based on the observations that have been made inestablishing the present invention, and although the invention is notlimited thereto, it is thought that such (pre-existing) proteins may inparticular (be able to) bind to the C-terminal end of such ISV's (which,in full sized conventional 4-chain monoclonal antibody as well as in the“heavy-chain only” antibodies that are found in Camelidae, are linked tothe rest of the antibody—i.e. to the CH1 region in conventionalmonoclonals and to the hinge region in Camelidae heavy chain antibodies,respectively—and thus in such full-sized antibodies may be shielded fromsuch protein interference).

This is confirmed by the findings made by the present inventors inestablishing the present invention (which findings are further describedherein) that certain (simple) modifications of ISV's at their C-terminalend may substantially reduce or essentially prevent such proteininterference. Accordingly, methods for modifying ISV's in this manner aswell as ISV's that have been modified in this manner form furtheraspects of the invention, as further described herein.

The present invention can in particular be used to reduce or avoidprotein interference and/or signals due to aspecific binding inimmunoassays that are performed on biological samples (such as blood orserum samples) obtained from a subject to whom a (biological) drug hasbeen administered (again, such samples are also referred to herein asthe “test sample” or “assay sample”). Some examples of this areimmunoassays that are used for characterization of drug disposition andof the formation of antibodies upon administration of a biological drugto a subject, such as those referred to in the “Guideline on theClinical Investigation of the Pharmacokinetics of Therapeutic Proteins”(document CHMP/EWP/89249/2004 dated Jan. 27, 2007) issued by theCommittee for Medicinal Products for Human Use (CHMP) of the EuropeanMedicines Agency (EMEA). As stated on pages 4 and 5 of this document:

-   -   “Several possible weaknesses have been identified and may result        in erroneous characterisation of drug disposition and of the        formation of antibodies. The following issues should be        considered [ . . . ]:    -   Immunoassay    -   Drug assay:    -   [ . . . ]    -   (iii) Interference by endogenous substances.    -   (iv) Interference by plasma components or anti-drug antibodies        binding to the analyte and inhibiting the complementary binding        to capture antibody.”

The invention can in particular be used in order to predict, reduce oravoid this type of interference in immunoassays that are used inanalyzing test samples/assay samples of biological fluids taken fromsubjects to whom ISV's (and in particular Nanobodies; or an ISV-basedbiological or Nanobody-based biological, as further defined herein) havebeen administered.

The invention can in particular be used in order to predict, reduce oravoid this type (aspecific) protein interference in immunoassays thatare used for characterization of drug disposition and/or for determiningthe formation of any ADA's (anti-drug) antibodies. In this respect,should be noted that generally in this specification and in the attachedclaims, when wording like “predicting, reduce or avoiding proteininterference” is used, this does not only include predicting, reduce oravoiding such protein interference per se, but also generallypredicting, reduce or avoiding the occurrence of aspecific signals inimmunoassays (such as those in which (aspecific) signals associated withprotein interference may occur, for example in ADA assays), and inparticular predicting, reduce or avoiding, in such immunoassays, theoccurrence of aspecific signals that, when they are observed in such anassay, are usually attributed to, associated with and/or taken as a signof (aspecific) protein interference. In this respect, it shouldgenerally be noted that, as mentioned herein, the present invention isnot specifically limited to any causation, explanation, hypothesis ormechanism.

In one specific but non-limiting aspect, the invention can be used topredict, avoid or reduce such protein interference in “anti-drugantibody” or “ADA” assays that are performed on samples (i.e., “testsamples”) of biological fluids taken from subjects to whom ISV's (and inparticular Nanobodies; or an ISV-based biological or Nanobody-basedbiological, as further defined herein) have been administered.

In another specific but non-limiting aspect, the invention can be usedto predict, avoid or reduce such protein interference (and/or aspecificsignals usually associated with the same) in “anti-drug antibody” or“ADA” assays that are used to detect, measure and/or characterize thepresence of (any) anti-drug antibodies against one or more ISV's (and inparticular against Nanobodies; or an ISV-based biological orNanobody-based biological, as further defined herein). In particular,the invention can be used to predict, avoid or reduce such proteininterference in such “anti-drug antibody” or “ADA” assays that areperformed on samples (i.e., “test samples”) of biological fluids, andmore in particular on samples of biological fluids of that have beenobtained from a subject to whom one or more such ISV's or Nanobodies (oran ISV-based biological or Nanobody-based biological, as further definedherein) have been administered. For example, the invention can be usedto predict, avoid or reduce such protein interference in such “anti-drugantibody” or “ADA” assays that are used to detect, measure and/orcharacterize the presence of (any) anti-drug antibodies against the ISVor Nanobody (or an ISV-based biological or Nanobody-based biological, asfurther defined herein) that has been administered to the subject fromwhich the sample has been obtained (either in the context of a clinicaltrial and/or in the context of therapy).

Thus, in one specific, but non-limiting aspect, the invention can beused to predict, avoid or reduce such protein interference (and/oraspecific signals usually associated with the same) in biologicalsamples (i.e., “test samples”) obtained from a subject to whom one ormore such ISV's or Nanobodies (or an ISV-based biological orNanobody-based biological, as further defined herein) have beenadministered, wherein said samples as suitable for and/or intended foruse in an immunological assay, such as an ADA assay. As mentioned, sucha biological sample may be blood (including whole blood, serum orplasma), ocular fluid, bronchoalveolar fluid/BALF, cerebrospinal fluidor any other suitable biological fluid or sample that is suitable foruse in an immunoassay, and in particular an ADA assay.

In one specific, but non-limiting aspect, such a test sample may havebeen obtained from a subject that has been subjected to multipleadministrations (for example at least 1 to 3 separate administrationsover a period of at least 10 days, such as at least one month or longer)and/or to chronic treatment (i.e. treatment during at least 10 days suchas at least one month) with an ISV, Nanobody, an ISV-based biological(as further defined herein) or Nanobody-based biological (as furtherdefined herein). Such an ISV, Nanobody, ISV-based biological orNanobody-based biological may for example have been administered to saidsubject in the context of therapy or in the context of a clinical trial.

In one specific, but non-limiting aspect, such a test sample may havebeen obtained from a subject to which a ISV, Nanobody, ISV-basedbiological or Nanobody-based biological has been administered that has(and/or has been provided with) an increased half-life (as definedherein, and compared to a monovalent ISV), for example a half-life of atleast 1 day, preferably at least 3 days, more preferably at least 7days, such as at least 10 days in the subject to which the same is/hasbeen administered.

For example and without limitation, such an ISV, Nanobody, ISV-basedbiological or Nanobody-based biological may have been provided with anincreased half-life by functionalization and/or by including in theconstruct a moiety or binding unit that increases the half-life of theconstruct. Examples of such functionalization, moieties or binding unitswill be clear to the skilled person and may for example be as describedherein, and for example may include pegylation, fusion to serum albumin,or fusion to a peptide or binding unit that can bind to a serum proteinsuch as serum albumin. Such a serum-albumin binding peptide or bindingdomain may be any suitable serum-albumin binding peptide or bindingdomain capable of increasing the half-life of the construct (compared tothe same construct without the serum-albumin binding peptide or bindingdomain), and may in particular be serum albumin binding peptides asdescribed in WO 2008/068280 by applicant (and in particular WO2009/127691 and the non-prepublished U.S. application 61/301,819, bothby applicant), or a serum-albumin binding ISV (such as a serum-albuminbinding Nanobody; for example Alb-1 or a humanized version of Alb-1 suchas Alb-8, for which reference is for example made to WO 06/122787).

Thus, in one specific but non-limiting aspect, such a biological samplemay have been obtained from a subject to which an ISV, Nanobody,ISV-based biological or Nanobody-based biological has been administeredthat comprises a (human) serum albumin-binding binding peptide orbinding domain.

As already mentioned above, in one non-limiting aspect, the inventiongenerally relates to a method that can be used to predict whether agiven ISV or Nanobody (or ISV-based or Nanobody-based drug) will giverise to (or has high or increased tendency to give rise to) proteininterference (as further described herein) in an immunoassay (i.e. aftersaid ISV has been administered to a subject, a sample of a biologicalfluid has been obtained from said subject, and said biological fluid issubjected to an immunoassay as further described herein), said methodcomprising performing an immunoassay that at least comprises the stepsof:

-   -   (i) contacting said ISV or Nanobody (or ISV-based or        Nanobody-based drug) with an antibody that has been obtained        from a human subject and that has been selected, generated        and/or isolated based on its ability to recognize and/or bind to        the C-terminal end of an ISV or Nanobody (the “analytical        antibody”); and    -   (ii) determining whether said ISV or Nanobody (or ISV-based or        Nanobody-based drug) is bound by said antibody in said        immunoassay.

Again, as mentioned herein, an ISV as described herein may in particulareither be a Nanobody or an(other) ISV (i.e. other than a Nanobody) thatis a VH domain or that comprises a VH domain; and is preferably aNanobody.

Also, any protein or polypeptide that comprises an ISV (such as anISV-based drug) preferably has said (or at least one) such ISV at itsC-terminal end. Again, said ISV may in particular either be a Nanobodyor an(other) ISV (i.e. other than a Nanobody) that is a VH domain orthat comprises a VH domain; and is preferably a Nanobody.

In an alternative embodiment, which is also further described herein,instead of the aforementioned antibody obtained from a human subject,the monoclonal antibody referred to herein as “21-4-3” (or “21-4” forshort, see SEQ ID NO's 35 and 36 for the VH and VL sequences) may beused. 21-4 was generated using hybridoma technology starting from amouse immunized with the Nanobody construct of SEQ ID NO:98 in WO2006/122825, as further described in Example 7, and a hybridoma cellline (called “ABH0015”) expressing 21-4 has been deposited on Jun. 4,2012 with the BCCM, Ghent, Belgium, under accession number LMBP-9680-CB.Monoclonal 21-4 has been shown to recognize the C-terminus the Nanobodyconstruct of SEQ ID NO:98 in WO 2006/122825, which C-terminal endconsists of a Nanobody (humanized V_(HH)) raised against Von WillebrandFactor (vWF). 21-4 was originally raised as analytical reagent for usein detecting the protein Nanobodies (n particular, the Nanobodyconstruct of SEQ ID NO:98 in WO 2006/122825) in (serum) samples;surprisingly, it has now been found that 21-4 can also be used in orderto predict whether an ISV has a tendency to undergo aspecific proteininterference (more so than some other, comparable (mouse) monoclonalsraised against the Nanobody construct of SEQ ID NO:98 in WO 2006/122825or against other Nanobodies).

In particular, it has been found that if measuring the binding of 21-4to an ISV (or to protein or polypeptide containing an ISV at itsC-terminal end, or similar protein or polypeptide as mentioned herein)gives an RU value of less than 500 (after adjusting the measured RUvalue for the molecular weight to the protein, according to the formula[RU measured]/[MW of the protein]×10⁶) when determined according to theprotocol set out in Example 9, that said ISV or protein will likely nothave a tendency to undergo protein interference (within the confidenceprovided by the data set out in the Examples below). For the purposes ofthe above formula, MW may be calculated as the sum of all the MW's ofall the amino acid residues present in the ISV.

Accordingly, any ISV, protein or polypeptide described herein preferablyhas such an RU value for binding by 21-4 of less than 500 (determinedaccording to the protocol set out in Example 9, and after adjusting themeasured RU value for the molecular weight of the ISV or protein usedaccording to the formula set out above).

Thus, this aspect of the invention generally relates to a method thatcan be used to predict whether a given ISV or Nanobody (or ISV-based orNanobody-based drug) will give rise to (or has high or increasedtendency to give rise to) protein interference (as further describedherein) in an immunoassay (i.e. after said ISV has been administered toa subject, a sample of a biological fluid has been obtained from saidsubject, and said biological fluid is subjected to an immunoassay asfurther described herein), said method comprising performing animmunoassay that at least comprises the steps of:

-   -   (i) contacting said ISV or Nanobody (or ISV-based or        Nanobody-based drug) with the monoclonal antibody 21-4 (i.e.        used as the “analytical antibody”); and    -   (ii) determining whether said ISV or Nanobody (or ISV-based or        Nanobody-based drug) is bound by the monoclonal antibody 21-4 in        said immunoassay.

Said method may in particular be performed using BiaCore or a similartechnique, and more in particular using the protocol set out in Example9. As mentioned herein, when the binding of the ISV or ISV-based drug inthis protocol shows an RU value of less than 500 (after adjusting themeasured RU value for the molecular weight to the protein, according tothe formula [RU measured]/[MW of the protein]×10⁶), said ISV orISV-based protein will likely not be bound by any interference factor(s)present in the blood or serum of a human being and/or will likely nothave a tendency to undergo aspecific protein interference in an ADAassay (i.e. within the degrees of confidence set out in the experimentalpart below).

Again, as mentioned herein, an ISV as described herein may in particulareither be a Nanobody or an(other) ISV (i.e. other than a Nanobody) thatis a VH domain or that comprises a VH domain; and is preferably aNanobody.

Also, any protein or polypeptide that comprises an ISV (such as anISV-based drug) preferably has said (or at least one) such ISV at itsC-terminal end. Again, said ISV may in particular either be a Nanobodyor an(other) ISV (i.e. other than a Nanobody) that is a VH domain orthat comprises a VH domain; and is preferably a Nanobody.

As also mentioned herein, the above method using 21-4 can also be usedto determine whether an ISV or protein or polypeptide that comprises aISV is bound by (or has a tendency to be bound by) interferencefactor(s) that are present in the blood or serum of a human being.

Also, as mentioned herein, it is envisaged that said method using 21-4can also be used to predict whether any protein or polypeptide (such asan antibody fragment or ScFv) that has a VH domain at its C-terminal endwill bound by (or has a tendency to be bound by) interference factor(s)that are present in the blood or serum of a human being and/or has atendency to undergo protein interference in an ADA assay.

In addition to 21-4, it is envisaged that an antibody or antibodyfragment (such as a suitable Fab fragment) that contains the heavy chainand light chain variable domains of 21-4 (see SEQ ID NO's: 35 and 36,respectively) or even only the CDR sequences of 21-4 (suitably graftedinto other suitable VH and VK frameworks) may also be used in themethods described herein.

As further described herein, the invention can in particular be used topredict whether a given ISV or Nanobody (or ISV-based or Nanobody-baseddrug) will give rise to protein interference (as further describedherein) in an immunoassay that is an ADA assay. Said ADA assay may forexample be an ADA assay for detecting or measuring ADA's against ISV'sgenerally, and may in particular be an ADA assay for detecting ormeasuring ADA's against the ISV used in steps (i) and (ii) above.

In one particular preferred but non-limiting aspect, the invention canbe used to predict whether a given ISV or Nanobody (or ISV-based orNanobody-based drug) will give rise to protein interference (as furtherdescribed herein) in an immunoassay (and in particular, in an ADA assay)that involves the use of such an ISV. Again, said ADA assay may forexample be an ADA assay for detecting or measuring ADA's against ISV'sgenerally, and may in particular be an ADA assay for detecting ormeasuring ADA's against the ISV used in steps (i) and (ii) above.

In an even more particular but non-limiting aspect, the invention can beused to predict whether a given ISV or Nanobody (or ISV-based orNanobody-based drug) will give rise to protein interference (as furtherdescribed herein) in an immunoassay (and in particular, in an ADA assay)that involves the use of such an ISV. For example, such an immunoassaymay be an ADA assay (i.e. involving the ISV) that is performed todetermine or measure whether any ADA's against said ISV are present inthe sample that is tested, wherein said sample is a sample of biologicalfluid (as described herein) that is obtained from a subject to whichsaid ISV has been administered (as further described herein). Forexample, as further mentioned herein, said sample (i.e., the “testsample”) may be a sample of (including whole blood, serum or plasma),ocular fluid, bronchoalveolar fluid/BALF, cerebrospinal fluid or anyother suitable biological fluid, and may in particular be a biologicalsample that is suitable for and/or intended for use in an immunologicalassay, such as an ADA assay.

As further described herein, in all these aspects (and the furtheraspects of the invention described herein), the invention can also beused to select ISV's that are not or less prone to such proteininterference in such immunoassays or ADA assays; as an assay or testthat can be used to test whether certain modification(s) to an ISV will(fully or partially) reduce its tendency to give rise to suchinterference in such immunoassays or ADA assays; and/or as an assay ortest that can be used to guide modification or improvement of an ISV soas to reduce its tendency to give rise to such protein interference insuch immunoassays or ADA assays.

As mentioned, step (i) of the method of the invention comprisescontacting said ISV or Nanobody (or ISV-based or Nanobody-based drug)with an antibody that has been obtained from a human subject and thathas been selected/isolated based on its ability to recognize and/or bindto the C-terminal end of an ISV or Nanobody (as further describedherein). In said step (i) of the method described herein, “said ISV orNanobody (or ISV-based or Nanobody-based drug)” is used as the antigenin the immunoassay (i.e. as the substance to be detected). Also, in saidstep (i), the “antibody that has been obtained from a human subject andthat has been selected/isolated based on its ability to recognize and/orbind to the C-terminal end of an ISV or Nanobody” is used as theanalytical reagent (i.e. in the same way as other antibodies are used inimmunoassays to detect the presence of an antigen to which they aredirected).

As already mentioned, and in order to better understand the inventiondescribed herein, it should be noted that, in step (i), the ISV willusually be used as the “antigen” (i.e., as the compound to be detected),and the “analytical antibody” will be used as the analytical agent(i.e., as a means to detect whether a given ISV binds or not,respectively; and thus has a high or increased risk of giving rise toprotein interference or not, respectively). For example, when step (i)is performed in an ELISA format, the “antibody/analytical agent” willusually be bound to the carrier (i.e., to the ELISA plate) and the ISVwill be (present in) the sample to be tested.

By contrast, it should be noted that in ADA assays for detecting ormeasuring ADA's against an ISV, the ISV is used as the “analyticalagent” (i.e., as the compound used to detect whether any ADA's arepresent), and the ADA's are the “antigen” (i.e., the compound to bedetected). Thus, in these assays, the ISV will usually/often be bound tothe carrier (such as the ELISA plate), whereas the ADA's (if any) willbe present in the sample that is subjected to the assay.

However, as already mentioned, it should generally be noted that theinvention is not limited to assays in which the “analytical antibody” isbound to the carrier. For example, in an alternative way of performingan assay according to the invention (as shown in Example 5), theanalytical antibody is instead used as a bridging agent and thus will bein solution rather than bound to the plate (although it is indirectlybound to the plate via the ISV that is coated on the plate). However,also in the specific (bridging) assay described in Example 5 (which is acompetitive assay) the analytical antibody is still used as theanalytical agent (i.e., to determine whether the ISV of interest bindsor not, respectively; and thus has a high or increased risk of givingrise to protein interference or not, respectively). It is also envisagedthat, based on the further disclosure herein, the skilled person will beable to design other assay formats in which the analytical antibody canbe used as an analytical agent in order to determine whether a given ISVcan bind or not, respectively; and thus has a high or increased risk ofgiving rise to protein interference or not).

The “analytical antibody” used in step (i) may be a polyclonal ormonoclonal antibody.

When the analytical antibody is a polyclonal antibody, it may forexample be a polyclonal antibody (preparation) that has beenobtained/purified/isolated from a biological sample obtained of a humansubject (such as blood, plasma, B-cells or another suitable biologicalsample or fluid from which polyclonal antibodies can be suitablyisolated). This may for example be a suitable biological sample that hasbeen obtained of a human subject to which at least one ISV (such as theISV used in step (i), but this is not required or essential) has beenadministered, but may also be (and preferably is) a suitable biologicalsample from a human subject which has never received or been treatedwith an ISV. What is more important is that the polyclonal antibody hasbeen obtained from said biological sample by a method that involves atleast one affinity step using an affinity matrix or column that carriesan ISV as the affinity moiety (and one or more further steps forobtaining/purifying/isolating polyclonal antibodies known per se). Forexample, the polyclonal antibody may have been obtained from such abiological sample by means of affinity chromatography using an affinitycolumn that carries an ISV, as for example described in Example 2. Thismay for example be performed using well known techniques forimmunoaffinity chromatography for isolating antibodies from a biologicalsample, using an affinity matrix that carries an ISV as the antigen.Such techniques are generally known in the art and suitable examplesthereof will be clear to the skilled person based on the disclosureherein.

Such a polyclonal antibody (preparation) may in particular be an IgG (orIgG fraction).

For example, it may be a polyclonal antibody that has been obtainedmeans of a method that involves (immuno)affinity chromatography,performed on a sample of biological fluid obtained from a human subject,using as the antigen bound to the affinity matrix an ISV (and inparticular a Nanobody, such as a VHH, humanized and/orsequence-optimized VHH or a camelized VH, such as a camelized human VH)that does not contain a C-terminal tag (i.e., of which the C-terminusends with the amino acid sequence VTVSS (SEQ ID NO:33)). In particular,the ISV used as the antigen bound to the affinity matrix may be ahumanized or sequence-optimized VHH (or alternatively a correspondingcamelized human VH) of which the C-terminus ends with the amino acidsequence VTVSS (SEQ ID NO:33). In one specific, but non-limiting aspect,the ISV used as the antigen bound to the affinity matrix may be ahumanized or sequence-optimized VHH that, as a result of suchhumanization or sequence-optimization, comprises a proline (P) residueat position 14 where the corresponding “naïve” VHH comprises an alanine(A) at position 14 (in other words, the ISV used as the antigen is ahumanized version of a VHH that naturally comprises an alanine atposition 14, which alanine residue, as a result of the humanizationand/or sequence optimization, has been replaced with a proline (P)residue). The ISV used as the antigen may also comprise one or moreother amino acid substitutions as a result of such humanization orsequence optimization, for example generally described in WO 08/020079or WO 09/138519.

Some specific examples of ISVs that can be used as the antigen togenerate/isolate the “analytical antibody” used in the invention aregiven in SEQ ID NO's: 1 and 2.

Again, the method used to obtain the polyclonal antibody may, inaddition to the (immune)affinity steps, also comprise one or morefurther steps for isolating/purifying a polyclonal antibody from thebiological sample (performed either before or after the affinity steps).Again, such steps and techniques for performing them will be clear tothe skilled person.

Thus, in one aspect, the invention comprises a method as furtherdescribed herein that comprises steps (i) and (ii) described herein, inwhich the “analytical antibody” (i.e., the antibody that has beenobtained from a human subject and that has been selected/isolated basedon its ability to recognize and/or bind to the C-terminal end of an ISVor Nanobody) has been obtained from a biological sample obtained from ahuman subject (wherein said biological sample is a sample that issuitable for use in a method for generating/isolating an antibody fromsaid sample) using a method that comprises at least one affinity step(such as a step of affinity chromatography, such as immunoaffinitychromatography) in which an ISV (and preferably a Nanobody) is used asan antigen, and preferably an ISV is used as an antigen that comprisesthe amino acid sequence VTVSS (SEQ ID NO:33) as the C-terminal sequence,and more preferably a humanized and/or sequence optimized Nanobody isused as the antigen that comprises the amino acid sequence VTVSS (SEQ IDNO:33) as the C-terminal sequence, and even more preferably a humanizedand/or sequence optimized Nanobody is used as the antigen that comprisesthe amino acid sequence VTVSS (SEQ ID NO:33) as the C-terminal sequenceand that comprises a proline residue at position 14, such as a Nanobodythat comprises the amino acid sequence VTVSS (SEQ ID NO:33) as theC-terminal sequence and that comprises a proline residue at position 14that has been introduced into the Nanobody as a result of saidhumanization and/or sequence-optimization (for example, to replace analanine residue that naturally occurs at said position in the VHH thathas been humanized and/or sequence optimized).

The above ISV's can also be used in methods to isolate monoclonalantibodies (again starting from a suitable biological sample obtainedfrom a human being) that are suitable for use in the invention as the“analytical antibody”.

For example, such a monoclonal antibody may be obtained starting fromblood, B-cells or another suitable sample or material for isolatingantibodies, may be selected based on its ability to recognize and/orbind to (the C-terminal end of) an ISV or Nanobody (in which, again, theISV(s) used as the antigen during screening and/or selection ispreferably as described in the preceding paragraphs, including thepreferences stated for such ISV/antigen). Such screening and selectionmay be performed in any suitable manner, for example by using B-cellselection and/or expansion techniques essentially the same or suitablysimilar to the B-cell selection techniques described in EP 0 488 470, WO92/02551, EP 1 633 787, WO 01/55216, WO 02/26829, WO 04/051268, WO04/102198 or WO 04/106377 or techniques similar to the Nanoclonetechnique described in WO 06/079372 (but using human B-cells rather thancamelid B-cells).

Once one or more B-cells have been identified/isolated that express asuitable antibody, said antibody may be isolated, expressed and/orproduced in any suitable manner. For example, said B-cell(s) may beimmortalized as hybridomas producing the desired antibody/antibodies(using techniques well known per se for generating hybridomas startingfrom selected B-cells), and said antibody/antibodies may then beisolated from (the culture supernatant of) said hybridoma(s), againusing suitable techniques well established in the art and described invarious handbooks and manuals, and also described and/or referred to inthe patent publications mentioned in the preceding paragraph.

Alternatively, said B-cell(s) may be expanded using B-cell expansiontechniques known per se, and the antibody/antibodies may be isolatedfrom (the culture supernatant of) said expanded B-cell(s). Again, thismay be performed using suitable techniques well established in the artand described in various handbooks and manuals, and also describedand/or referred to in the patent publications mentioned in the precedingparagraphs.

In yet another alternative, DNA encoding the antibody/antibodies ofinterest may be obtained (e.g., by amplification) from said B-cell(s) orother suitable cells, either directly (for example using suitablesingle-cell PCR cloning techniques) or after suitable expansion of thedesired B-cell(s). Said DNA may then be suitably expressed in a suitablehost cell or host organism to provide the desired antibody/antibodies.Again, this may be performed using suitable techniques well establishedin the art and described in various handbooks and manuals, and alsodescribed and/or referred to in the patent publications mentioned in thepreceding paragraphs.

It is also possible to generate monoclonal antibodies that are suitablefor use as the “analytical antibody” by a method that involvesrepertoire cloning (starting from a suitable sample obtained from ahuman subject) and screening the cloned repertoire for antibodies thatbind to the ISV used as antigen (in which, again, the ISV(s) used as theantigen during screening and/or selection is preferably as described inthe preceding paragraphs, including the preferences stated for suchISV/antigen). Methods for repertoire cloning and various techniques fordisplaying cloned repertoires for selection and screening (such as phagedisplay, ribosome display and yeast display) will be clear to theskilled person, and are for example described in EP 0 589877, EP 0 774511, WO 90/14430 and EP 0368 684) as well as various handbooks on thesubject.

Generally, the biological sample that is used as a starting point forobtaining the (polyclonal or monoclonal) analytical antibody may be anysuitable sample (i.e. suitable as a starting material for obtaining apolyclonal or monoclonal antibody, respectively) obtained from anysuitable human subject. In one specific but non-limiting aspect, such asample may for example have been obtained from a woman, and inparticular a post-menopausal woman. Thus, in one specific butnon-limiting aspect, the analytical antibody used in steps (i) and (ii)above has been obtained starting from a biological sample that has beenobtained/derived from a post-menopausal woman (or has been derived froman antibody that has been obtained/derived from a post-menopausalwoman).

Also, the biological sample that is used as a starting point forobtaining the (polyclonal or monoclonal) analytical antibody may beobtained from a subject to whom an ISV has previously been administered(for example, as part of a clinical trial or therapeutically), but ispreferably obtained from a subject to whom no ISV has previously beenadministered.

However, it should be noted that the invention is not particularlylimited to the source of the analytical antibody/antibodies used, and ithas proven possible in some cases, using the techniques describedherein, to obtain (generate, isolate) other suitable analyticalantibodies from other sources, including commercially available humanblood or plasma (and even blood, plasma or B-cells from other species ofmammals or primates, such as from baboon or cynomolgus monkey).

As mentioned above, the (polyclonal or monoclonal) analytical antibodyused in steps (i) and (ii) should be such that it is capable ofrecognizing or binding to the C-terminal end of an ISV or Nanobody, andis most preferably selected and/or isolated based on this ability tobind to the C-terminal end of an ISV or Nanobody.

As can be seen from FIG. 2, when the ISV is based on or derived from aVH or VHH domain, the C-terminal end of an ISV comprises the amino acidsequence VTVSS (SEQ ID NO:33), and accordingly the analytical antibodyshould be capable of recognizing any ISV that has the amino acidsequence VTVSS (SEQ ID NO:33) at its C-terminal end. As can be furtherseen from FIG. 2, (at least some of the amino acid residues in) thesequence VTVSS (SEQ ID NO:33) is part of a putative epitope on the ISVthat also includes, among other residues, the amino acid residue atposition 14 (and the amino acid residues next/close to the same in theamino acid sequence, such as positions 11, 13 and 15) and may alsocomprise the amino acid residue at position 83 (and the amino acidresidues next/close to the same in the amino acid sequence, such aspositions 82, 82a, 82b and 84) and/or the amino acid residue at position108 (and the amino acid residues next/close to the same in the aminoacid sequence, such as positions 107. Position 109 is the first V of theC-terminal VTVSS (SEQ ID NO:33) sequence and it has been shown that forexample position 110 may have an influence on protein interference aswell). This is also collectively referred to herein as the “C-terminalregion”, it being understood that this C-terminal region at leastcomprises the C-terminal sequence VTVSS (SEQ ID NO:33) and the aminoacid residue at position 14, and may also comprise the amino acidresidues at positions 83 and 108, and possibly also the amino acidresidues at positions 13, 15, 82b, 83, 84 and 107.

As already mentioned, and again without being limited to any hypothesisor explanation, in a full-sized 4-chain monoclonal antibody, or in afull-sized heavy chain only antibody such as those present in Camelidae,the C-terminal end of a VH or VHH domain is linked to the rest of theantibody—i.e. to the CH1 region in conventional monoclonals or to thehinge region in Camelidae heavy chain antibodies, respectively—and thusin such full-sized antibodies may be shielded from such proteininterference) and/or covered by the VH/VL interaction (in conventional4-chain antibodies) so that this “C-terminal region” and is thereforeusually not solvent-exposed and/or accessible as an interaction site forproteins that are present in the blood, serum or body of a person towhich such an ISV is administered. However, if an ISV or Nanobody isused per se (i.e. without being linked to any other part of anantibody), or if an ISV-based drug or Nanobody-based drug is used thatcarries an ISV or Nanobody at its C-terminal end, this C-terminalepitope is available for (aspecific) interaction with other proteins,and again without being limited to any hypothesis or explanation, it isassumed that this C-terminal region may now be accessible to undergo an(aspecific) protein interaction with one or more proteins that arepre-existing in the “test sample” (for example, one or more IgG's) to betested and that this may cause protein interference and/or aspecificsignals in the immunoassays (and in particular in ADA assays).

As mentioned, the methods described herein can be used to predict,reduce or avoid such protein interaction, and can also be used as a toolto guide modification to the ISV, Nanobody, ISV-based drug orNanobody-based drug so as to provide the same with a (partially orpreferably essentially fully) reduced tendency to give rise to suchprotein interference.

As will be clear from the preceding paragraph, and again without beinglimited to any hypothesis or explanation, it is in particular expected(and part of the teaching of the present invention) that (certain)modifications to the “C-terminal region” will alter (and preferablyreduce) the tendency of an ISV to undergo such aspecific proteininteraction, and this is also what is observed experimentally (see forexample the experimental results presented in Examples 1C and 3 below).

Based on this, and again without being limited to any hypothesis orexplanation, the present invention also teaches certain modificationsthat can be introduced for this purpose in the C-terminal region of anISV, Nanobody, ISV-based drug or Nanobody-based drug (of which the(potential) effectiveness can be tested using the methods describedherein). Also, based on the teaching herein, it is envisaged that theskilled person will be able to choose, design or propose other(candidate) modifications to the C-terminal region that could beintroduced for this purpose (and of which the (potential) effectivenesscan again be tested using the methods described herein).

Returning to the analytical antibody used in the invention, this ispreferably a (polyclonal or monoclonal) antibody that recognizes theC-terminal region (as defined above) of an ISV, and in particular butwithout limitation the C-terminal region of a Nanobody.

For example, in one specific but non-limiting aspect, the “analyticalantibody” may be a polyclonal or monoclonal that recognizes (and/or iscapable of binding to, and in particular of specific binding to) theC-terminal region of an ISV or Nanobody of which the C-terminal end ofthe sequence ends with VTVSS (SEQ ID NO:33), but does not recognize(and/or is not capable of specific binding to) the C-terminal region ofan ISV or Nanobody (which may be a different ISV but is preferably thesame ISV) when there are one or more further amino acid residues (suchas 1 to 5 amino acid residues, or alternatively a small peptide sequenceor even another polypeptide or protein) linked to the C-terminal VTVSS(SEQ ID NO:33).

In another, more specific but still non-limiting aspect, the “analyticalantibody” may be a polyclonal or monoclonal that recognizes (and/or iscapable of binding to, and in particular of specific binding to) theC-terminal region of an ISV or Nanobody of which the C-terminal end ofthe sequence ends with VTVSS (SEQ ID NO:33) and in which position 14 isan amino acid that does not naturally occur at position 14 and/or hasbeen modified compared to the amino acid that naturally occurs atposition 14 (for example as a result of humanization, camelizationand/or sequence optimization), but that does not recognize (and/or isnot capable of specific binding to) the C-terminal region of an ISV orNanobody (which may be a different ISV but is preferably the same ISV)in which there are one or more further amino acid residues (such as 1 to5 amino acid residues, or alternatively a small peptide sequence or evenanother polypeptide or protein) linked to the C-terminal VTVSS (SEQ IDNO:33); and/or in which position 14 is an amino acid that naturallyoccurs at position 14 (for example alanine or, when the ISV naturallycontains a proline at position 14, proline).

For example, the “analytical antibody” may also be a polyclonal ormonoclonal that recognizes (and/or is capable of binding to, and inparticular of specific binding to) the C-terminal region of an ISV orNanobody of which the C-terminal end of the sequence ends with VTVSS(SEQ ID NO:33) and in which position 14 is proline (and in particularwhen position 14 has been modified to proline, for example as a resultof humanization, camelization and/or sequence optimization), but doesnot recognize the C-terminal region of an ISV or Nanobody (which may bea different ISV but is preferably the same ISV) in which there are oneor more further amino acid residues (such as 1 to 5 amino acid residues,or alternatively a small peptide sequence or even another polypeptide orprotein) linked to the C-terminal VTVSS (SEQ ID NO:33); and/or in whichposition 14 is alanine.

The “analytical antibody” may also be a polyclonal or monoclonal thatrecognizes (and/or is capable of binding to, and in particular ofspecific binding to) the C-terminal region of an ISV or Nanobody ofwhich the C-terminal end of the sequence ends with VTVSS (SEQ ID NO:33)and in which position 14 is proline (in particular where a prolineresidue naturally occurs at said position in said ISV), but does notrecognize the C-terminal region of an ISV or Nanobody (which may be adifferent ISV but is preferably the same ISV) in which there are one ormore further amino acid residues (such as 1 to 5 amino acid residues, oralternatively a small peptide sequence or even another polypeptide orprotein) linked to the C-terminal VTVSS (SEQ ID NO:33) in which position14 is still a (naturally occurring or unmodified) proline.

The “analytical antibody” may also for example be a polyclonal ormonoclonal that recognizes (the C-terminal region of) the sequence ofthe ISV called “Nb 3.4” herein (SEQ ID NO: 5) but does not recognize(the C-terminal region of) the sequence of the ISV called “Nb 3.1”herein (SEQ ID NO: 3) and/or (and preferably and) does not recognize thesequence of the ISV called “Nb 3.2” herein (SEQ ID NO: 4).

For the above purpose, whether an “analytical antibody” does (or doesnot) recognize an ISV or Nanobody (and/or is or is not capable of(specifically) binding to an ISV or Nanobody) can be determined usingany suitable binding assay (such as Biacore), but may also be determinedusing either the BIACORE assay described in example 3 or an ADA assaysuch as the ADA bridging/competition assay described in Example 5 (Seealso FIG. 1A to 1C and in particular FIG. 1B).

Suitable formats/techniques for performing such an assay will be clearto the skilled person based on the disclosure herein, and for exampleinclude (without limitation):

-   -   A colorimetric assay such as ELISA with analytical antibody        coated directly or indirectly to the plate and detection of        bound ISV with monoclonal or polyclonal anti-ISV antibody. Other        useful alternative technologies for this setup include but are        not limited to electrochemiluminescence (the MSD platform),        Fluorescence (DELFIA, GYROS), and other methods that rely on        secondary detection of the bound ISV.    -   A Surface Plasmon Resonance (such as BIACORE) or other real-time        biosensor method (i.e. other than using SPR) with directly or        indirectly immobilized analytical antibody and monitoring the        binding of subsequently injected/administered ISV. These methods        do not need further detection of the bound ISV. A representative        method for performing this type is assay is described in Example        3.    -   Analyzing the competitive behavior of the ISV in a bridging        assay (ADA assay) using the analytical antibody instead of ADA        containing biological fluid. For the bridging assay one can make        use of different technologies such as ELISA, the MSD platform.        Representative methods for performing this type are        schematically shown in FIGS. 1A to 1C and one specific example        of this kind of assay is also described in Example 5.    -   Any chromatographic method in which the analytical antibody is        immobilized on the chromatographic matrix and specific        capturing/isolation of ISV from a solution.

Once a suitable analytical antibody has been obtained using one of themethods described herein or in one of the examples (or a methodessentially equivalent to the same), said analytical antibody can beused to determine whether a given ISV or Nanobody (or ISV-based orNanobody-based drug) will give rise to (or has high or increasedtendency to give rise to) protein interference (as defined herein), i.e.by performing steps (i) and (ii) described above. As already describedherein, this generally involves contacting said ISV, Nanobody, ISV-baseddrug or Nanobody-based drug with the analytical antibody and determiningwhether said ISV, Nanobody, ISV-based drug or Nanobody-based drug isrecognized by (and/or is bound by, and in particular specifically boundby) said analytical antibody (and in particular whether the C-terminalregion of said ISV or Nanobody or of any ISV or Nanobody that forms theC-terminal end of said ISV-based drug or Nanobody-based drug isrecognized by said analytical antibody).

This can generally be performed using any suitable technique fordetermining whether an antigen (in the case, the ISV, Nanobody,ISV-based drug or Nanobody-based drug) is bound by an antibody, andsuitable (immune) as say techniques will be clear to the skilled person.Some non-limiting examples are suitable ELISA techniques (including forexample sandwich ELISA's); in which, depending on the ELISA format used(as will be clear to the skilled person), either the analytical antibodyor the ISV may be coated on the plate and either the analytical antibodyor the ISV may be detectably labeled. Other techniques may for exampleinvolve the use of a BIAcore instrument (in which again either theanalytical antibody or the ISV may be coated on the chip, see forexample Example 3). Another alternative may be a competitive bridgingassay (as for example exemplified in Example 5), in which the ability istested of the ISV to compete with another ISV, Nanobody, ISV-based drugor Nanobody-based drug that is known to be bound by the analyticalantibody (or visa versa). These and other suitable techniques fordetermining whether a given ISV, Nanobody, ISV-based drug orNanobody-based drug is (specifically) bound or recognized by theanalytical antibody will be clear to the skilled person based on thedisclosure herein.

It will also be clear, based on the disclosure herein, that the presentinvention (and in particular the analytical antibody used in the presentinvention) can be used to determine whether or not a given ISV,Nanobody, ISV-based drug or Nanobody-based drug contains an interactionsite (such as an interaction site present at or within the C-terminalregion, and/or of which the C-terminal region forms part) that iscapable of undergoing an (aspecific) protein interaction with one ormore proteins or other components that may be present in a biologicalsample (i.e., a “test sample”) obtained from a subject that is to besubjected to an immunoassay such as an ADA assay (in particular, an ADAassay for determining the presence of any ADA's against the ISV,Nanobody, ISV-based drug or Nanobody-based drug). Thus, when an ISV,Nanobody, ISV-based drug or Nanobody-based drug is recognized by theanalytical antibody used in the invention, it is very likely that saidISV, Nanobody, ISV-based drug or Nanobody-based drug contains such an(accessible or exposed) interaction site, and thus will have a tendencyto give rise to such protein interference (as defined herein) when it isused in such an immunoassay or ADA assay for testing the test sample. Aswill be clear to the skilled person, this is something that shouldpreferably be avoided, either by selecting/using another ISV, Nanobody,ISV-based drug or Nanobody-based drug if possible, or by modifying theISV, Nanobody, ISV-based drug or Nanobody-based such that its tendencyto such protein interference will be substantially reduced oressentially removed (again, this can be tested using the method andanalytical antibody disclosed herein).

As will also be clear to the skilled person based on the disclosureherein, such a modification may for example comprise making one or moremodifications (such as amino acid insertions, additions, deletions orsubstitutions) to the interaction site on the ISV, Nanobody, ISV-baseddrug or Nanobody-based drug, such that its ability to undergo an(aspecific) protein interaction with one or more proteins or othercomponents that may be present in a test sample will be reduced orremoved. Again, this can be performed by limited trial and error byintroducing one or more modifications and then testing whether thisability has been reduced or not, again using the method and analyticalantibody disclosed herein. For example, one or more such modificationsmay be introduced, and then the ability of the modified ISV to bind tothe analytical antibody may be compared to that of theoriginal/unmodified ISV. Alternatively, using a competitive bridgingformat (as for example exemplified in Example 5), or using BIAcore (seefor example Example 3), the ability of the modified ISV to (still)compete with the original ISV for binding to the analytical antibody maybe determined.

Again, and although the invention is not limited to any hypothesis orexplanation, based on the experimental evidence that is set out in theexamples below, the inventors have found that this interaction site islikely located at/near the C-terminal region (as defined herein) or thatsaid interaction site forms part of the C-terminal region (or that theC-terminal region forms part of this interaction site). This is forexample based at least in part on the observation that, if an ISV has atendency to give rise to such protein interference and has VTVSS (SEQ IDNO:33) as the amino acid residues at its C-terminal end, that attachingeither a limited number of amino acid residues (such as 1 to 10, forexample 1 to 5, such as 1, 2, 3, 4 or 5), or alternatively a tag oranother peptide, protein or other moiety to this C-terminal end willusually substantially reduce or essentially remove said tendency. Insome cases, it has been found that even adding 1, 2 or 3 amino acidresidues to the C-terminal VTVSS (SEQ ID NO:33) (which may be anysuitable amino acid(s) or combination of amino acids, which may each beindependently chosen from any naturally occurring amino acids such asthose listed in Table A-2 on page 64 of WO 09/138519, for example andwithout limitation from alanine, glycine, valine, leucine or isoleucine)may already substantially reduce or essentially remove said tendency.This is also in part based on the observation that in some cases, wherea VHH naturally contains an alanine residue at position 14 (which asmentioned forms part of the C-terminal region; see FIG. 2), thenaturally occurring VHH often does not have (or has a low) tendency togive rise to such protein interference, whereas a corresponding VHH inwhich said alanine at position 14 has been replaced with a prolineresidue (for example, for the purposes of humanization orsequence-optimization) can as a result have an increased tendency togive rise to such protein interference (i.e. compared to the VHH withalanine at position 14).

In one aspect, the invention relates to a VHH, a Nanobody (as definedherein, and in particular a humanized VHH or a camelized VH, such as acamelized human VH) or another ISV (or ISV-based drug or Nanobody-baseddrug with a VHH, Nanobody or other ISV at its C-terminal end) that hasbeen modified (for example, by introducing one or more amino acidsubstitutions, additions or deletions), and in particular modified inthe C-terminal regions (such as by one or more amino acid substitutionsor additions in the C-terminal region), such that (i) it has asubstantially reduced tendency (such as at least a statisticallyrelevant reduced tendency) to give rise to protein interference (asdefined herein); and/or such that (ii) it has, in the method of theinvention described herein (such as in the specific assay described inExample 3 or 5), substantially reduced ability to be bound by ananalytical antibody as described herein (such as the polyclonal antibodydescribed in Example 2 and used in Examples 3 and 5), in both casespreferably compared to the same VHH, Nanobody or ISV but without themodifications.

Thus, in one aspect, the invention relates to a VHH, a Nanobody (asdefined herein, and in particular a humanized VHH or a camelized VH,such as a camelized human VH) or another ISV (or ISV-based drug orNanobody-based drug with a VHH, Nanobody or other ISV at its C-terminalend) that is a VHH or VH domain (i.e. an ISV that is a VH domain orderived from a VH domain) and/or that has been based on or has beenderived from (the amino acid sequence of) a VHH or VH domain, which VHH,Nanobody or ISV comprises the amino acid sequence VTVSS(X)_(n) (SEQ IDNO:34) at its C-terminal end, in which n is 1 to 10, preferably 1 to 5,such as 1, 2, 3, 4 or 5 (and preferably 1 or 2, such as 1), and in whicheach X is an (preferably naturally occurring) amino acid residue that isindependently chosen (and preferably independently chosen from the groupconsisting of alanine (A), glycine (G), valine (V), leucine (L) orisoleucine (I); however, as can be seen from the data presented below,other (preferably naturally occurring) amino acid residues orcombinations of the aforementioned preferred amino acid residues withother amino acid residues (such as serine, proline, threonine and/orlysine) may also be used). Preferably, said VHH, Nanobody or ISV withthe amino acid sequence VTVSS(X)_(n) (SEQ ID NO:34) at its C-terminalend is such that (i) it has a substantially reduced tendency (such as atleast a statistically relevant reduced tendency) to give rise to proteininterference (as defined herein); and/or such that (ii) it has, in themethod of the invention described herein (such as in the specific assaydescribed in Example 3 or 5), substantially reduced ability to be boundby an analytical antibody as described herein (such as the polyclonalantibody described in Example 2), in both cases preferably compared tothe same VHH, Nanobody or ISV but with the amino acid sequence VTVSS(SEQ ID NO:33) at its C-terminal end. Reference is for example made tothe assay and data presented in Example 3.

The aforementioned VHH's, Nanobodies or (other) ISVs are preferably suchthat they have an RU value for binding by 21-4 of less than 500(determined according to the protocol set out in Example 9, and afteradjusting the measured RU value for the molecu It should also be notedthat, any time that reference is made in the description herein or inthe claims to any C-terminal sequence VTVSS(X)_(n) (including any of theaspects (a) to (p) above, that according to one specific aspect of theinvention, none of the amino acids X is a cysteine residue.

For example, in some preferred aspects, the C-terminal end of the ISV orISV-containing construct (when this C-terminal end is a VH-derived ISV,VHH or Nanobody) may be:

-   (a) VTVSS(X)_(n), in which n=1 and X=Ala;-   (b) VTVSS(X)_(n), in which n=2 and each X=Ala;-   (c) VTVSS(X)_(n), in which n=3 and each X=Ala;-   (d) VTVSS(X)_(n), in which n=2 and at least one X=Ala (with the    remaining amino acid residue(s) X being independently chosen from    any naturally occurring amino acid but preferably being    independently chosen from Val, Leu and/or Ile);-   (e) VTVSS(X)_(n), in which n=3 and at least one X=Ala (with the    remaining amino acid residue(s) X being independently chosen from    any naturally occurring amino acid but preferably being    independently chosen from Val, Leu and/or Ile);-   (f) VTVSS(X)_(n), in which n=3 and at least two X=Ala (with the    remaining amino acid residue(s) X being independently chosen from    any naturally occurring amino acid but preferably being    independently chosen from Val, Leu and/or Ile);-   (g) VTVSS(X)_(n), in which n=1 and X=Gly;-   (h) VTVSS(X)_(n), in which n=2 and each X=Gly;-   (i) VTVSS(X)_(n), in which n=3 and each X=Gly;-   (j) VTVSS(X)_(n), in which n=2 and at least one X=Gly (with the    remaining amino acid residue(s) X being independently chosen from    any naturally occurring amino acid but preferably being    independently chosen from Val, Leu and/or Ile);-   (k) VTVSS(X)_(n), in which n=3 and at least one X=Gly (with the    remaining amino acid residue(s) X being independently chosen from    any naturally occurring amino acid but preferably being    independently chosen from Val, Leu and/or Ile);-   (l) VTVSS(X)_(n), in which n=3 and at least two X=Gly (with the    remaining amino acid residue(s) X being independently chosen from    any naturally occurring amino acid but preferably being    independently chosen from Val, Leu and/or Ile);-   (m) VTVSS(X)_(n), in which n=2 and each X=Ala or Gly;-   (n) VTVSS(X)_(n), in which n=3 and each X=Ala or Gly;-   (o) VTVSS(X)_(n), in which n=3 and at least one X=Ala or Gly (with    the remaining amino acid residue(s) X being independently chosen    from any naturally occurring amino acid but preferably being    independently chosen from Val, Leu and/or Ile); or-   (p) VTVSS(X)_(n), in which n=3 and at least two X=Ala or Gly (with    the remaining amino acid residue(s) X being independently chosen    from any naturally occurring amino acid but preferably being    independently chosen from Val, Leu and/or Ile);    with aspects (a), (b), (c), (g), (h), (i), (m) and (n) being    particularly preferred, with aspects in which n=1 or 2 being    preferred and aspects in which n=1 being particularly preferred.

It should also be noted that, any time that reference is made in thedescription herein or in the claims to any C-terminal sequenceVTVSS(X)_(n) (including any of the aspects (a) to (p) above, thataccording to one specific aspect of the invention, none of the aminoacids X is a cysteine residue.

Thus, in one preferred aspect, the invention relates to animmunoglobulin single variable domain (ISV), which is either a Nanobodyor an(other) ISV that comprises a VH sequence or is derived from a VHsequence (with Nanobodies being preferred) which has a C-terminal end ofthe sequence VTVSS(X)_(n), in which n=1 and X=Ala (or a protein orpolypeptide which contains such an ISV (and preferably such a Nanobody)at its C-terminal end).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=2 and each X=Ala (or a protein or polypeptidewhich contains such an ISV (and preferably such a Nanobody) at itsC-terminal end).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=2 and at least one X=Ala (with the remainingamino acid residue(s) X being independently chosen from any naturallyoccurring amino acid but preferably being independently chosen from Val,Leu and/or Ile) (or a protein or polypeptide which contains such an ISV(and preferably such a Nanobody) at its C-terminal end).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=3 and at least one X=Ala (with the remainingamino acid residue(s) X being independently chosen from any naturallyoccurring amino acid but preferably being independently chosen from Val,Leu and/or Ile) (or a protein or polypeptide which contains such an ISV(and preferably such a Nanobody) at its C-terminal end).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=3 and at least two X=Ala (with the remainingamino acid residue(s) X being independently chosen from any naturallyoccurring amino acid but preferably being independently chosen from Val,Leu and/or Ile) (or a protein or polypeptide which contains such an ISV(and preferably such a Nanobody) at its C-terminal end).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=3 and each X=Ala (or a protein or polypeptidewhich contains such an ISV (and preferably such a Nanobody) at itsC-terminal end).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=1 and X=Gly (or a protein or polypeptide whichcontains such an ISV (and preferably such a Nanobody) at its C-terminalend).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=2 and each X=Gly (or a protein or polypeptidewhich contains such an ISV (and preferably such a Nanobody) at itsC-terminal end).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=3 and each X=Gly (or a protein or polypeptidewhich contains such an ISV (and preferably such a Nanobody) at itsC-terminal end).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=2 and at least one X=Gly (with the remainingamino acid residue(s) X being independently chosen from any naturallyoccurring amino acid but preferably being independently chosen from Val,Leu and/or Ile) (or a protein or polypeptide which contains such an ISV(and preferably such a Nanobody) at its C-terminal end).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=3 and at least one X=Gly (with the remainingamino acid residue(s) X being independently chosen from any naturallyoccurring amino acid but preferably being independently chosen from Val,Leu and/or Ile) (or a protein or polypeptide which contains such an ISV(and preferably such a Nanobody) at its C-terminal end).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=3 and at least two X=Gly (with the remainingamino acid residue(s) X being independently chosen from any naturallyoccurring amino acid but preferably being independently chosen from Val,Leu and/or Ile) (or a protein or polypeptide which contains such an ISV(and preferably such a Nanobody) at its C-terminal end).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=2 and each X=Ala or Gly (or a protein orpolypeptide which contains such an ISV (and preferably such a Nanobody)at its C-terminal end).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=3 and each X=Ala or Gly (or a protein orpolypeptide which contains such an ISV (and preferably such a Nanobody)at its C-terminal end).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=3 and at least one X=Ala or Gly (with theremaining amino acid residue(s) X being independently chosen from anynaturally occurring amino acid but preferably being independently chosenfrom Val, Leu and/or Be) (or a protein or polypeptide which containssuch an ISV (and preferably such a Nanobody) at its C-terminal end). or

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=3 and at least two X=Ala or Gly (with theremaining amino acid residue(s) X being independently chosen from anynaturally occurring amino acid but preferably being independently chosenfrom Val, Leu and/or Be) (or a protein or polypeptide which containssuch an ISV (and preferably such a Nanobody) at its C-terminal end).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which n=1, 2 or 3 in which each X=Ala or Gly.

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which:

-   -   n=1, 2 or 3 in which each X=Ala or Gly; or    -   n=2 or 3 in which all but one X=Ala or Gly (with the remaining        amino acid residue X being independently chosen from any        naturally occurring amino acid but preferably being        independently chosen from Val, Leu and/or Ile)        or a protein or polypeptide which contains such an ISV (and        preferably such a Nanobody) at its C-terminal end).

In another preferred aspect, the invention relates to an immunoglobulinsingle variable domain (ISV), which is either a Nanobody or an(other)ISV that comprises a VH sequence or is derived from a VH sequence (withNanobodies being preferred) which has a C-terminal end of the sequenceVTVSS(X)_(n), in which:

-   -   n=1, 2 or 3 in which each X=Ala or Gly; or    -   n=2 or 3 in which at least one X=Ala or Gly (with the remaining        amino acid residue X being independently chosen from any        naturally occurring amino acid but preferably being        independently chosen from Val, Leu and/or Ile);    -   n=2 or 3 in which all but one X=Ala or Gly (with the remaining        amino acid residue X being independently chosen from any        naturally occurring amino acid but preferably being        independently chosen from Val, Leu and/or Ile);        or a protein or polypeptide which contains such an ISV (and        preferably such a Nanobody) at its C-terminal end.

In the above aspects, with said (other) “ISV that comprises a VHsequence or is derived from a VH sequence” is meant any ISV thatcomprises a VH sequence or that is derived from a VH sequence and thatis not a Nanobody (i.e. not a VHH, humanized VHH or camelized VH). Forexample, such (other) ISV may for example be a VH-based (single) domainantibody, VH-based dAb™, or VH-based microbody (see WO 00/29004).

Again, it should be noted that, any time that one of the ISV's referredto herein has a C-terminal sequence VTVSS(X)_(n) (including withoutlimitation in ISV's referred to in the preceding aspects) that accordingto one specific aspect of the invention, none of the amino acids X inthe sequence VTVSS(X)_(n) is a cysteine residue.

As further described herein, any such protein or polypeptide may forexample be a construct that contains two or more ISV's (such as two ormore Nanobodies), optionally linked via one or more suitable linkers.Thus, for example, such a construct may be a bivalent, trivalent,tetravalent or pentavalent construct (such as a bivalent, trivalent,tetravalent or pentavalent Nanobody construct), and may for example be abivalent, trivalent, tetravalent or pentavalent construct (such as abivalent, trivalent, tetravalent or pentavalent Nanobody construct) thatis bispecific, trispecific or biparatopic construct (including forexample monospecific, bispecific or biparatopic constructs that also canbind to serum albumin (preferred) or another serum protein for half-lifeextension).

Again, the Nanobodies, ISVs and proteins/polypeptides according to eachof the aspects described above are preferably such that they have an RUvalue for binding by 21-4 of less than 500 (determined according to theprotocol set out in Example 9, and after adjusting the measured RU valuefor the molecular weight of the ISV or protein used according to theformula set out above).

As mentioned herein, it is also envisaged that the invention may also beapplied to other proteins or polypeptides (and in particular antibodyfragments such as Fab fragments or other proteins or polypeptides basedon antibody fragments, such as ScFv's) that have a VH-domain at theirC-terminal end. Thus, in another aspect, the invention relates to such aprotein or polypeptide (such as an ScFv) that has a VH domain at itsC-terminal end with the amino acid sequence VTVSS(X)_(n) (SEQ ID NO:34)at its C-terminal end, in which n is 1 to 10, preferably 1 to 5, such as1, 2, 3, 4 or 5, and in which each X is an (preferably naturallyoccurring) amino acid residue that is independently chosen (andpreferably independently chosen from the group consisting of alanine(A), glycine (G), valine (V), leucine (L) or isoleucine (I). Again,according to some specific aspects, said C-terminal end may be accordingto any of (a) to (p) above, and preferably according to one of (a), (b),(c), (g), (h), (i), (m) or (n), with n being 1, 2 or 3 and preferably 1or 2.

Again, such proteins or polypeptides are preferably such that they havean RU value for binding by 21-4 of less than 500 (determined accordingto the protocol set out in Example 9, and after adjusting the measuredRU value for the molecular weight of the ISV or protein used accordingto the formula set out above). Also, again, according to one specificaspect of this aspect of the invention, none of the amino acids X in theC-terminal sequence VTVSS(X)_(n) is a cysteine residue.

The invention further relates to a pharmaceutical composition thatcomprises an ISV (and preferably a therapeutic ISV) or a protein orpolypeptide comprising at least one ISV (and preferably at least onetherapeutic ISV), wherein said ISV, protein or polypeptide is as furtherdescribed herein (i.e. an ISV, protein or polypeptide according to oneor more of the aspects described herein, and in particular according toone or more of the aspects described on the preceding pages; and more inparticular an ISV, protein or polypeptide that has a C-terminalend/sequence that is according to one or more of the aspects describedherein), and at least one suitable carrier, diluent or excipient (i.e.suitable for pharmaceutical use), and optionally one or more furtheractive substances. Such compositions, carriers, diluents or excipientscan for example be as described in WO 08/020079 for pharmaceuticalcompositions that comprise a Nanobody or a protein or polypeptide thatcomprises at least one Nanobody (and as already mentioned, according tothe present invention, the ISV is also preferably a Nanobody).

The invention further relates to an ISV or a protein or polypeptidecomprising at least one ISV for use in therapy of a disease in a humanbeing (e.g. a patient in need of such therapy), wherein said ISV,protein or polypeptide is as further described herein (i.e. an ISV,protein or polypeptide according to one or more of the aspects describedherein, and in particular according to one or more of the aspectsdescribed on the preceding pages; and more in particular an ISV, proteinor polypeptide that has a C-terminal end/sequence that is according toone or more of the aspects described herein).

The invention further relates to the use of an ISV or a protein orpolypeptide comprising at least one ISV in the preparation of apharmaceutical composition, wherein said ISV, protein or polypeptide isas further described herein (i.e. an ISV, protein or polypeptideaccording to one or more of the aspects described herein, and inparticular according to one or more of the aspects described on thepreceding pages; and more in particular an ISV, protein or polypeptidethat has a C-terminal end/sequence that is according to one or more ofthe aspects described herein).

The invention further relates to a method of treatment which comprisesadministering to a human subject (e.g to a patient in need of suchtreatment) an ISV or a protein or polypeptide comprising at least oneISV in the preparation of a pharmaceutical composition, wherein saidISV, protein or polypeptide is as further described herein (i.e. an ISV,protein or polypeptide according to one or more of the aspects describedherein, and in particular according to one or more of the aspectsdescribed on the preceding pages; and more in particular an ISV, proteinor polypeptide that has a C-terminal end/sequence that is according toone or more of the aspects described herein); or a pharmaceuticalcomposition (as described above) that comprises at least one such ISV,protein or polypeptide.

With respect to the above, it will be clear that the therapeutic use ofthe ISV's, proteins and polypeptides described herein are a veryimportant aspect of the invention, as such therapeutic use (or theclinical development of such ISV's, proteins and polypeptides for suchtherapeutic use) may involve the use of ADA assays to determine whethersaid ISV, protein or polypeptide is immunogenic (i.e. can give rise toADA's when administered to a human subject). In this respect, it willalso be clear that concerns about possible immunogenicity will inparticular have to be addressed when a therapeutic is either used forlonger periods of time (for during weeks, months or years), and/or has ahalf-life (preferably expressed as t½-beta) in a human subject of atleast 3 days, such as at least one week, and up to 10 days or more.

Thus, according to one specific aspect of the invention, a ISV, protein,polypeptide or pharmaceutical composition as described herein isintended for treatment of a chronic disease in a human being, and/orsuch ISV, protein, polypeptide as described herein is intended to bepresent in the circulation of the subject (i.e. at pharmacologicallyactive levels) to which it is administered (i.e. at a therapeuticallyactive dose) for at least a period of one week, preferably at least twoweeks, such as at least a months; and/or such ISV, protein, polypeptideas described herein is such that it has a half-life (preferablyexpressed as t½-beta) in a human subject of at least 3 days, such as atleast one week, and up to 10 days or more; and/or such an ISV, protein,polypeptide or pharmaceutical composition as described herein isintended to be administered to a human being as two or more doses thatare administered over a period of at least 3 days, such as at least oneweek, for example at least two weeks or at least one month, or evenlonger (i.e. at least 3 months, at least 6 months or at least one year),or even chronically administered.

The invention further relates to a method for (substantially) reducingor essentially removing the tendency of an ISV, a Nanobody or anISV-based drug or a Nanobody-based drug to give rise to proteininterference, said method comprising at least the steps of:

-   -   optionally determining the tendency of the ISV, Nanobody,        ISV-based drug or Nanobody-based drug to give rise to protein        interference, using a method that at least comprises steps (i)        and (ii) as referred to herein;    -   modifying said ISV, Nanobody, ISV-based drug or Nanobody-based        drug by introducing one or more one or more amino acid        substitutions, additions or deletions in said ISV or Nanobody,        or in the C-terminal ISV or Nanobody (if any) of the ISV-based        drug or Nanobody-based drug; and in particular by introducing        one or more amino acid substitutions or additions in the        C-terminal region of said ISV or Nanobody, or in the C-terminal        region of the C-terminal ISV or Nanobody (if any) of the        ISV-based drug or Nanobody-based drug, for example by adding to        the C-terminal end of the sequence 1 to 10, such as 1 to 5, such        as 1, 2, 3, 4 or 5 amino acid residues each independently chosen        from any naturally occurring amino acids (such as those listed        in Table A-2 on page 64 of WO 09/138519, for example and without        limitation from alanine, glycine, valine, leucine or        isoleucine);    -   determining the tendency of the so modified ISV, Nanobody,        ISV-based drug or Nanobody-based drug to give rise to protein        interference, using a method that at least comprises steps (i)        and (ii) as referred to herein; optionally in a manner that        allows the tendency of the so modified ISV, Nanobody, ISV-based        drug or Nanobody-based drug to give rise to protein interference        to be compared to the tendency of the original ISV, Nanobody,        ISV-based drug or Nanobody-based drug to give rise to protein        interference (including, without limitation, comparing them in a        competition assay for binding to the analytical antibody as        described herein). Alternatively, the method described herein        that involves the use of 21-4 may be used.

The invention will now be further described by means of the followingnon-limiting preferred aspects, examples and figures, in which:

FIG. 1A to 1C schematically shows some non-limiting examples of ADAassay formats. Some representative but non-limiting protocols forperforming these assays are mentioned in Example 4.

FIG. 2 schematically shows a representative 3D structure of an ISV, suchas a Nanobody.

FIG. 3 is a binding curve (obtained using the BIACORE assay described inExample 3) showing the binding of NB's 3.4 to 3.9 (SEQ ID NO's. 5 to 10)to the immobilized polyclonal antibody obtained in Example 2.

FIG. 4 is a binding curve (obtained using the BIACORE assay described inExample 3) showing the binding of NB's 3.4, 3.11, 3.12 and 3.13 (SEQ IDNO's: 5, 12, 13 and 14) to the immobilized polyclonal antibody obtainedin Example 2.

FIG. 5 is a binding curve (obtained using the BIACORE assay described inExample 3) showing the binding of NB's 3.4, 3.14 and 3.15 (SEQ ID NO's:5, 15 and 16) to the immobilized polyclonal antibody obtained in Example2.

FIG. 6 is a binding curve (obtained using the BIACORE assay described inExample 3) showing the binding of NB's 3.1, 3.2 and 3.4 (SEQ ID NO's: 3,4 and 5) to the immobilized polyclonal antibody obtained in Example 2.

FIG. 7 is a binding curve (obtained using the BIACORE assay described inExample 3) showing the binding of NB's 4.1 and 4.2 (SEQ ID NO's: 17 and18) to the immobilized polyclonal antibody obtained in Example 2.

FIG. 8 is a binding curve (obtained using the BIACORE assay described inExample 3) showing the binding of NB's 6.1, 6.2, 6.4 and 6.5 (SEQ IDNO's 19 to 22) to the immobilized polyclonal antibody obtained inExample 2.

FIG. 9 gives is a Table showing the sequences used in Example 8 (SEQ IDNO's: 37 to 89) and setting out the corresponding reference sequence.

The sequences referred to in the present description and claims arelisted in Table A below (SEQ ID NO's: 1 to 37) and in FIG. 9 (SEQ IDNO's: 38 to 89).

TABLE A SEQ ID Name NO: Sequence ISV Ex. 1/2- 1EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGIKSSGDSTRYAGSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAKSRVSRTGLYTYDNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMGWFRQAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAASAIGSGALRRFE YDYSGQGTLVTVSS Alt. ISV 2EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLVTVSS >Nb3.1 3EVQLVESGGGLVQAGGSLRLSCAASRSIGRLDRMGWYRHRTGEPRELVATITGGSSINYGDFVKGRFTISIDNAKNTVYLQMNNLKPEDTAVYYCNFNKYVTSRDTWGQGTQVTVSS >Nb3.2 4EVQLVESGGGLVQAGGSLRLSCAASRSIGRLDRMGWYRHRTGEPRELVATITGGSSINYGDFVKGRFTISIDNAKNTVYLQMNNLKPEDTAVYYCNFNKYVTSRDTWGQGTQVTVSSAAAEQKLISEED LNGAAHHHHHH >Nb3.4 5EVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSS >Nb3.5 6HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSAA >Nb3.6 7HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSA >Nb3.7 8HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSG >Nb3.8 9HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSGG >Nb3.9 10HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSGGG >Nb3.10 11HHHHHHEVQLVESGGGLVQAGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSS >Nb3.11 12HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLKPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSS >Nb3.12 13HHHHHHEVQLVESGGGLVQAGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTQVTVSS >Nb3.13 14HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTQVTVSS >Nb3.14 15HHHHHHEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVQVSS >Nb3.15 16HHHHHHEVQLVESGGGSVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSS >Nb4.1 17EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSS >Nb4.2 18EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGG GSRDWDFDVFGGGTPVGG >Nb6.1 19EVQLVESGGGLVQPGGSLRLSCIASGLPFSTKSMGWFRQAPGKEREFVARISPGGTSRYYGDFVKGRFAISRDNAKNTTWLQMNSLKAEDTAVYYCASGERSTYIGSNYYRTNEYDYWGTGTQVTVSSAAAEQKLISEEDLNGAAHHHHHH >Nb6.2 20EVQLVESGGGLVQPGGSLRLSCIASGLPFSTKSMGWFRQAPGKEREFVARISPGGTSRYYGDFVKGRFAISRDNAKNTTWLQMNSLKAEDTAVYYCASGERSTYIGSNYYRTNEYDYWGTGTQVTVSS >Nb6.4 21EVQLLESGGGLVQPGGSLRLSCAASGLPFSTKSMGWFRQAPGKGREFVSRISPGGTSRYYGDFVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASGERSTYIGSNYYRTNEYDYWGQGTLVTVSSA AAEQKLISEEDLNGAAHHHHHH >Nb6.522 EVQLLESGGGLVQPGGSLRLSCAASGLPFSTKSMGWFRQAPGKGREFVSRISPGGTSRYYGDFVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASGERSTYIGSNYYRTNEYDYWGQGTLVTVSS Example 1C: 23HHHHHHEVQLVESGGGLVQAGGSLRLSCAASGRTFNNYAMG wildtypeWFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGT QVTVSS Example 1C: 24HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMG (A14P)WFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGT QVTVSS Example 1C: 25HHHHHHEVQLVESGGGLVQAGGSLRLSCAASGRTFNNYAMG (K83R)WFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLRPEDTAVYTCAASAIGSGALRRFEYDYSGQGT QVTVSS Example 1C: 26HHHHHHEVQLVESGGGLVQAGGSLRLSCAASGRTFNNYAMG (Q108L)WFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNAKNTLYLQMNSLKPEDTAVYTCAASAIGSGALRRFEYDYSGQGT LVTVSS Example 1C: 27HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMG (A14P, K83R,WFRRAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNA Q108L)KNTLYLQMNSLRPEDTAVYTCAASAIGSGALRRFEYDYSGQGT LVTVSS Example 1c: 28HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMG (A14P, R39Q,WFRQAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNA K83R, T91Y,KNTLYLQMNSLRPEDTAVYYCAASAIGSGALRRFEYDYSGQGT Q108L) LVTVSS Example 1C: 29HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMG (A14P, R39Q,WFRQAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNA K83R, T91Y,KNTLYLQMNSLRPEDTAVYYCAASAIGSGALRRFEYDYSGQGT Q108L)-1A LVTVSSAExample 1C: 30 HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGRTFNNYAMG (A14P ,R39Q,WFRQAPGKEREFVAAITRSGVRSGVSAIYGDSVKDRFTISRDNA K83R, T91Y,KNTLYLQMNSLRPEDTAVYYCAASAIGSGALRRFEYDYSGQGT Q108L)-3A LVTVSSAAA Nb3.1631 DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSS Nb3.17 32DVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASRSIGRLDRMGWYRHRPGEPRELVATITGGSSINYGDSVKGRFTISIDNSKNTVYLQMNSLRPEDTAVYYCNFNKYVTSRDTWGQGTLVTVSSA C-terminal 33 VTVSS sequenceC-terminal 34 VTVSS(X)_(n) sequence 21-4-3, IGH 35QIQLVQSGPELKKPGETVKISCKASGYTFTAYSMHWVKQAPG consensusKGLKWMGWINTVTGEPAYADDFKGRFAFSLETSASTAYLQISSLKNEDTATYFCTRGLIHFYYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKK IVPRDC 21-4-3-IGK 36DIQMTQTPSSLSASLGGRVTITCKASQDIHNFISWYQHKPGKV consensusPRLIIHDTSTLQPGIPSRFSGSGSGRDYSFSITNLEPEDIATYYCLHYDNLLRSFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

EXPERIMENTAL PART Example 1: Generation of a Polyclonal AnalyticalAntibody

A polyclonal antibody (IgG fraction) that can be used as the “analyticalantibody” was generated as follows:

A. Identification of Suitable Plasma Samples for Isolating thePolyclonal Antibody

Twenty plasma samples from healthy individuals that were never treatedwith an ISV were evaluated for presence of antibodies against ISV thatcan be used as the analytical antibody in the invention.

The ISV that was initially used in this Example was SEQ ID NO: 1.Subsequently, to confirm that the interaction is not specific for thisparticular ISV, but is an aspecific protein-protein interaction that mayoccur with a number of ISV's, the assays below were repeated with otherISV's (see paragraph C) below). As an alternative for SEQ ID NO:1, forexample SEQ ID NO:2 may also be used.

The assay used was an ECL (Electrochemiluminescence) based bridgingassay that used biotinylated ISV (a biotinylated variant of SEQ ID NO:1)to capture and sulfo-tagged ISV to detect anti-drug antibodies. Asimilar format is also used for performing ADA assays. Biotinylation andsulfo-tagging of the ISV was done using standard coupling chemistry onprimary amines using Sulfo-NHS-LC-Biotin (Pierce) and Sulfo-tagNHS-Ester (MSD), respectively according to the manufacturer'sinstructions. The plasma samples were diluted ⅕ in PBS/0.1% casein andwere incubated for 30 minutes at 37° C., 600 RPM in 96 wellpolypropylene plates. The samples (50 μL) were then diluted ⅓ in 1:1mixture (100 μL) of 2 μg/ml biotinylated and 2 μg/ml sulfo-tagged ISV(SEQ ID NO:1) and incubated for 1 hour at RT, 600 RPM. MSD MA®96-wellStandard Streptavidin plates were blocked with 150 μL/well Superblock®T20 for 1 hour at RT, then washed 3 times with PBS/0.05% Tween20 (=washbuffer). Sample/1:1 mix (biotinylated and sulfo-tagged ISV (SEQ ID NO:1)(50.0 μL) was transferred from the polypropylene plate to the MSD plateand incubated for 1 hour at RT, 600 rpm. Plates were washed three timesprior to addition of 2× Read Buffer (MSD) (150 μL/well) and reading theECL units (ECLU) on an MSD instrument (Sector Imager 2400 reader).Samples were screened as positive or negative using the screeningcut-point determined during method validation. The screening cut-pointwas calculated based on the background values of 118 individual plasmasamples from healthy individuals that were never treated with an ISV,using appropriate statistical analysis as recommended by the guidelinesfor ADA assay development (Shankar, 2008). A non-parametric assessmentwas used and the cut-off value was calculated based on the 95^(th)percentile, after exclusion of outliers.

Six plasma samples were clearly scored as positive: IHuP#002-001-ABL-01, IHuP #002-001-ABL-08, IHuP #002-001-ABL-10, IHuP#002-001-ABL-15, IHuP #002-001-ABL-19 and IHuP #002-001-ABL-20 (TableI).

These samples were further analyzed in a drug displacement set-up(confirmatory assay) to confirm the specificity of the positivescreening outcome (Table II). Therefore, the samples were diluted ⅕ inPBS/0.1% casein containing 12.5 μg/mL ISV (SEQ ID NO:1) and wereincubated for 30 minutes at 37° C., 600 RPM in 96 well polypropyleneplates. The samples (50 μL) are then diluted ⅓ in 1:1 mixture (100 μL)of 2 μg/ml biotinylated and 2 μg/ml sulfo-tagged ISV (SEQ ID NO:1) andincubated for 1 hour at RT, 600 RPM. Subsequently, sample/1:1 mix(biotinylated and sulfo-tagged ISV) (50.0 μL) was transferred from thepolypropylene plate to the blocked MSD MA®96-well Standard Streptavidinplate as described above for the screening assay and incubated for 1hour at RT, 600 rpm. Plates were washed three times prior to addition of2×Read Buffer (MSD) (150 μL/well) and measuring ECL units (ECLU) on anMSD instrument (Sector Imager 2400 reader). Samples were confirmed astrue positives using the confirmatory cut-point determined during methodvalidation and was calculated on the ECL response of 118 individualplasma samples from healthy individuals that were never treated withISV, that were spiked with 50 μg/ml ISV (SEQ ID NO:1) using appropriatestatistical analysis as recommended by the guidelines for ADA assaydevelopment (Shankar, 2008). A minimal signal reduction of 50% wascalculated based on the 99% confidence interval.

Samples that were positive in the ECL based bridging assay and that wereconfirmed as positive in the drug displacement set-up assay wereselected as a source for generating the polyclonal antibody usingaffinity chromatography.

TABLE I screening results of 20 plasma samples in the ADA ISV assay.Sample ID ECLU screening assay IHuP#002-001-ABL-01 13081IHuP#002-001-ABL-02 56 IHuP#002-001-ABL-03 272 IHuP#002-001-ABL-04 125IHuP#002-001-ABL-05 70 IHuP#002-001-ABL-06 99 IHuP#002-001-ABL-07 170IHuP#002-001-ABL-08 659358 IHuP#002-001-ABL-09 798 IHuP#002-001-ABL-101101 IHuP#002-001-ABL-11 83 IHuP#002-001-ABL-12 72 IHuP#002-001-ABL-13403 IHuP#002-001-ABL-14 62 IHuP#002-001-ABL-15 1141 IHuP#002-001-ABL-16159 IHuP#002-001-ABL-17 72 IHuP#002-001-ABL-18 170 IHuP#002-001-ABL-194503 IHuP#002-001-ABL-20 8243

TABLE II Confirmation of positively screened plasma samples in theconfirmatory assay. A confirmatory cut-point of 50% was used forevaluation of the results. One sample was not confirmed as a truepositive sample ECLU Plasma ECLU screening confirmatory % signal sampleID assay: plasma assay: plasma inhibition IHuP#002-001-ABL-01 13081 68595 IHuP#002-001-ABL-08 659358 169410 74 IHuP#002-001-ABL-10 1101 582 47IHuP#002-001-ABL-15 1141 467 59 IHuP#002-001-ABL-19 4503 1531 66IHuP#002-001-ABL-20 8243 1450 82

A further three serum samples from individuals that not have beentreated with an ISV were also evaluated using the ECL based bridgingassay described above and confirmed using the drug displacement set-upassay.

Two serum samples were clearly scored as positive in the ECL basedbridging assay: IHUS #B09032311A3 and IHUS #B09032311A20 (Table III).The 2 positively screened samples were further analyzed in the drugdisplacement set-up to confirm the specificity of the positive screeningoutcome.

TABLE III Screening and confirmatory results of 3 serum samples andcorresponding IgG purified fraction ECL ECL signal ECL signal signal ECLsignal screening confirmatory screening confirmatory assay: assay: %signal assay: assay: % signal Serum sample ID serum serum inhibition IgGIgG inhibition IHUS#B09032311A3 2388 286 88% 3716 370 90%IHUS#B09032311A20 19272 915 95% 31309 1160 96% IHUS#B09032311A1 62B. Generation of Purified Polyclonal IgG Fraction.

A polyclonal IgG was purified from the samples IHUS #B09032311A3 andIHUS #B09032311A20 (see above) using Protein G HP Spin Trap Columns (GEHealthcare) according to the manufacturer's instructions. In short,after removal of the storage solution form the column by centrifugation(30s at 100×g), the column was equilibrated by adding binding buffer (20mM sodium phosphate, pH 7.0). After centrifugation, the solutioncontaining the desired polyclonal was added (max 1 mg in 600 μl) andcolumn was incubated for 4 min while gently mixing. The column was thencentrifuged and washed 2× by successive addition of binding buffer (600μl) and centrifugation. After addition of 400 μl elution buffer (0.1 Mglycine-HCL, pH 2.7) and mixing by inversion, the antibody was eluted bycentrifugation in 30 μl neutralization buffer (1M Tris-HCL, pH 9.0).

In order to confirm that the IgG fraction thus obtained was involved inaspecific binding to the ISV(s), the purified IgG antibody was analyzedin the ECL based bridging assay described above and confirmed using thedrug displacement set-up assay used under A) above. In both samples(IHUS #B09032311A3 and IHUS #B09032311A20), purified IgG antibody wasconfirmed to be involved in the aspecific binding leading to a positivesignal in the assays (Table III). This confirmed that the purifiedpolyclonal IgG could be used as an “analytical antibody”, and it wasused as such in (the assays of) Examples 3 and 5.

C. Aspecific Binding to Other ISV's.

In order to determine whether the protein interference observed isspecific for a single ISV, and/or is specific for a particular region,epitope or antigenic determinant on ISV's, and/or for certain mutationsmade to wildtype ISV's (such as one or more humanizing mutations), theECL based bridging assay and the drug displacement set-up assay (both asdescribed under A) above, with SEQ ID NO: 1 being used as thesulfo-tagged ISV) were repeated using the plasma samples IHUS#B09032311A3, IHUS #B09032311A20 and IHUS #B09032311A1. As these plasmasamples contain the polyclonal “analytical” antibody isolated under B)above, this also provides information on the specificity, selectivityand epitope recognition of the polyclonal analytical antibody.

8 ISV's were tested (SEQ ID NO's 23 to 30, respectively—see Table Aabove), of which one was a wildtype VHH (SEQ ID NO: 23) and the other 7ISV's were humanized versions of the wildtype sequence with differenthumanizing substitutions. Two ISV's (SEQ ID NO's: 29 and 30) alsocontained additional amino acid residues at the C-terminus (1 and 3additional alanine residues, respectively).

The data are shown in Table IV. Without being limited to any explanationor hypothesis, it can be seen that changes to the C-terminal region (asdefined herein) can apparently strongly influence the extent to whichthe plasma samples used can give rise to protein interference. Forexample, it can be seen that adding one or three amino acid residues tothe C-terminus can strongly reduce the tendency for protein interferenceto arise (for example, only 18 and 13% reduction in the ECLU assay withsample IHUS #B09032311A3 for SEQ ID NO's: 29 and 30, compared to 90%reduction for SEQ ID NO: 28, the corresponding humanized variant withoutany amino acid residues added to the C-terminus). Similarly, introducinga proline residue at position 14 of the wildtype sequence can apparentlyalso strongly influence the extent to which the plasma samples used cangive rise to protein interference (for example, only 20% reduction inthe ECLU assay with sample IHUS #B09032311A3 for the wildtype sequenceof SEQ ID NO's: 23, compared to 91% reduction for SEQ ID NO: 24, thewildtype sequence with an A14P substitution). K83R and Q108L, which arealso substitutions close to the C-terminal region, also lead to someincrease in the tendency to give rise to protein interference, but notas much as the A14P substitution, and the total combined effect of theA14P+K83R+Q108L substitutions can be negated by adding one or more aminoacid residues to the C-terminus (compare again the data for SEQ ID NO's:29 and 30 with the data for the other humanized variants).

Based on this data, it was also concluded that apparently, thepolyclonal analytical antibody recognized the C-terminal region (asdefined herein) of ISV's generally. As can be seen from FIG. 2, position14 (and to a lesser degree positions 83 and 108) also form parts of theC-terminal region of an ISV (when the three-dimensional ternarystructure of an ISV is taken into account).

TABLE IV Evaluation of different Nanobody variants as competitor in theISV ADA assay using the analytical antibody. IHUS#B09032311IHUS#B09032311 IHUS#B09032311 Serum sample ID A3 A20 A1 ECLU inscreening assay (using SEQ ID NO: 1) 2217 18494 62 Nanobody Variant(right hand column mentions the humanizing substitutions and C-terminalECLU ECLU ECLU additions made compared to the confirmatory %confirmatory % confirmatory % wildtype sequence of SEQ ID NO:23 assayreduction assay reduction assay reduction SEQ ID NO: Wildtype VHH 177820 8682 53 60 4 23 SEQ ID NO: Wildtype VHH + 205 91 668 96 56 10 24 A14PSEQ ID NO: Wildtype VHH + 1403 37 6912 63 62 1 25 K83R SEQ ID NO:Wildtype VHH + 1533 31 6991 62 59 5 26 Q108L SEQ ID NO: Wildtype VHH +156 93 628 97 57 8 27 A14P + K83R + Q108L SEQ ID NO: Wildtype VHH + 22890 570 97 58 6 28 A14P + R39Q + K83R + T91Y + Q108L SEQ ID NO: WildtypeVHH + 1814 18 15087 18 60 3 29 A14P + R39Q + K83R +T91Y + Q108L + 1additional A at C-terminus (A114) SEQ ID NO: Wildtype VHH + 1933 1315244 18 62 0 30 A14P + R39Q + K83R + T91Y + Q108L + 3 A's at C-terminus(A114 + A115 + A116)

Example 2: Affinity Purification of Analytical Antibody

This Example describes two methods that can be used to isolate from abiological fluid from a human subject an analytical antibody that isable to recognize and/or bind the C-terminal end of an ISV. The antibodyis isolated from 4 different serum samples that were characterized inthat these induced a positive signal in an ADA assay according to thetest as described in Example 1.

Starting from serum samples, each of these protocols provide a purifiedpreparation of interference factor(s) that can be used as the analyticalantibody in the methods described herein. These methods can also moregenerally be used to purify the interference factor(s) for otherpurposes (for example, the interference factor(s) purified using theprotocols below were also used experimentally in Example 8 in order toshow that binding to an ISV or ISV-construct by monoclonal 21-4 ispredictive for binding of the same ISV or ISV-construct by interferencefactors, and thus by the tendency of said ISV or ISV-construct toundergo aspecific protein interference in an ADA assay).

Example 2A: Purification Using Protein A and Affinity Chromatography

In a first step, the IgG antibody fraction was enriched from the serumsamples using protein A affinity chromatography. Typical columns thatwere used for this enrichment included HiTrap MabselectSure andMabSelectXtra (GE Healthcare); PorosMabCapture A (Applied Biosystems).Purification of the IgG antibodies from the serum samples was performedin an automated and similar manner over all experiments. Chromatographicruns were performed on the AKTA purifier systems (GE Healthcare) andlogged in real-time using UNICORN protein purification software (GEHealthcare). Briefly, the serum sample was diluted 1:1 with D-PBS(Dulbecco's Phosphate-Buffered Saline) and 0.22 μm filtered beforeuploading on the column at a fixed flow rate of 0.5 mL/min. The columnwas washed to remove non-specific binding components over 5 columnvolumes using D-PBS at a flow rate of 0.5 mL/min. The IgG fraction waseluted by acidic elution, using 100 mM Glycine pH 2.6 buffer, and a flowrate of 0.5 mL/min. After elution, the fractions were neutralized using1.5 M Tris buffer pH 8.8. SDS-PAGE was run to confirm the isolation ofIgG antibodies in the elution.

In a second step, the interfering IgGs were further enriched by applyingthe protein A purified IgG fraction from the 4 different sera onto ISVcoupled affinity columns. More specifically, the interfering IgG werefurther enriched by binding to a column containing an ISV with sequenceof SEQ ID NO: 1. To this, the ISV was covalently linked to Sepharose 4fast flow (GE Healthcare) using the CNBr (Cyanogen bromide)-couplingmethod according to the manufacturer's procedure. The affinitypurification was performed in an automated and similar manner over allexperiments. Chromatographic runs were performed on the AKTA purifiersystems and logged in UNICORN. Briefly, the IgG enriched sample (up to10 mL loading volume) was uploaded on the column at a fixed flow rate of0.5 mL/min. The column was washed to remove non-specificbinding-components over 5 column volumes using D-PBS at a flow rate of0.5 mL/min. The ISV-binding components were eluted by acidic elution,using 100 mM Glycine pH 2.6 buffer, and a flow rate of 0.5 mL/min. Afterelution, the fractions were neutralized using 1.5 M Tris buffer pH 8.8.The fractions were analyzed using SDS-PAGE which confirmed the isolationof IgG antibodies in the elution (data not shown).

These fractions were pooled and used for further analyses such as thosedescribed in Example 3.

Example 2B: Purification Suing CaptureSelect™ Chromatography

Alternatively, interference factor(s) were recovered from plasma andpurified using the commercially available IgA binding affinity resinCaptureSelect hIgA™ (BAC BV), which is based on camelid-derivedheavy-chain only variable domains (VHH). The collected ‘IgA fraction’containing IgA together with interfering IgG was subsequently loadedonto a protein A column to remove the IgA fraction. The protein A columnwas processed according to generic IgG purification conditions (runningbuffer: PBS; elution buffer: 100 mM glycine pH=2.7; post elutionneutralization via 1M Tris). The interference factor was recovered fromthe Prot A elution in >95% yield.

In a variation to this method, another CaptureSelect affinity resin(CaptureSelect Alpha-1 Antitrypsin resin, a VHH based commerciallyavailable affinity resin, not targeting any antibody related proteins)was be used. This resin provided a high interference factor bindingefficacy and allowed for a selective 2 step elution: antitrypsin vianeutral pH elution using 2.0 M MgCl2, followed by the interferencefactor elution via an acidic step (0.1 M Glycine pH3.0, similar toprotein A/G elution conditions; neutralisation using 1.5M Tris). Thisone step purification yielded up to 15 μg interfering IgG1 per mL highinterference plasma, which is approximately 0.3% of the total IgGpresent. Optionally, the neutralised interference fraction can bedesalted and further purified via a Size Exclusion Column equilibratedin D-PBS.

Example 3: Influence of Different ISV Substitutions on the Tendency ofISV to Give Rise to Protein Interference

As mentioned in the description above, the present invention makesavailable certain assays and techniques which make it possible to makean assessment of whether or not a given ISV has a tendency to give riseto protein interference. These include the ECL based bridging assay andthe drug displacement set-up assay used in Example 1, as well as theBIACORE assay described in this Example 3 and the bridging/competitionADA assay described in the further Examples below.

As also mentioned in the description above, these assays can also beused to determine whether specific changes (such as amino aciddeletions, substitutions or additions) can influence (and preferablyreduce) the tendency of a given ISV to give rise to proteininterference. Some of these changes will be or become clear to theskilled person based on the disclosure herein and on the experimentaldata presented in Example 1 and this Example 3.

As already indicated by the data generated in Example 1, it appears thatcertain mutations in or close to the C-terminal region (as definedherein) of an ISV can (strongly) influence its tendency to give rise toprotein interference. For example, adding a few amino acid residues tothe C-terminus (such as 1 or 3 alanine residues) appears to stronglyreduce the tendency of an ISV to give rise to protein interference, andappears even to be able to negate the presence of other substitutions(for example, in or close to the C-terminal region) which appear toincrease the tendency to give rise to protein interference (for example,an A14P substitution).

In this Example 3, both the effect of other substitutions as well as theeffect of adding additional amino acids to the C-terminus wasinvestigated by comparing related ISV's with different substitutions,using the analytical polyclonal antibody generated in Example 2. Theanalysis was done by measuring the kinetics of interaction between eachof the ISV's investigated and the analytical polyclonal by means ofsurface Plasmon resonance (SPR) using the Biacore™ T100 biosensor fromGE Healthcare. The ISV tested in this Example 3 were those of SEQ IDNO's 3 to 22 (see Table A above and Table V below).

In a typical experiment, a polyclonal antibody solution was prepared at10 μg/ml in 10 mM NaOAc pH5.0. This polyclonal antibody was thenimmobilized on a CMS sensorchip using amine coupling by the EDC/NHSmethod (EDC═N-ethyl-N′-[3-diethylamino-propyl]-carbodiimide;NHS═N-hydroxysuccinimide) according to the manufacturer's procedure. Theamount immobilized gave approximately 2700 response units (RU). A fixedconcentration of 500 nM of ISV was then injected onto the surface for120 seconds at a flow rate of 45 μl per minute. Because no efficientregeneration buffer could be identified, the dissociation time waselongated to 2400 seconds. The signal obtained by injecting the ISV ontoa blank flow cell was subtracted from the signal obtained by injectingthe ISV onto the polyclonal antibody bound flow cell. The blank flowcell was activated/deactivated in a similar way as the flow cell for thepolyclonal antibody, but without adding protein. Also, a blank injection(HBS-EP+running buffer (HBS=Hepes Buffered Saline: GE Healthcare) wassubtracted to correct for possible baseline drift.

To examine the effect of adding amino acid residues to the C-terminus,the influence of adding 1 or 2 alanines and 1, 2 or 3 glycines wasinvestigated by comparing the binding of ISV with the differentadditions, using an analytical polyclonal antibody generated asdescribed in example 2. The ISV's generated and tested for this purposewere NB's 3.4 to 3.9 (SEQ ID NO's: 5 to 10).

As representative examples of the kind of data obtained, FIG. 3 showsthe binding of NB's 3.4 to 3.9 to the immobilized polyclonal antibody.Table V summarizes the results obtained.

TABLE V SEQ ID Position Position Position Position Binding** Clone ID NO113⁽¹⁾ 114⁽¹⁾ 115⁽¹⁾ 116⁽¹⁾ (RU) NB 3.4 5 S 75 NB 3.5 6 S A 9 NB 3.6 7 SA 8 A NB 3.7 8 S G 31 NB 3.8 9 S G G 13 NB 3.9 10 S G G G 13 **Bindingsignal obtained at the end of injection (=maximal RU signal) ⁽¹⁾In thisnumbering, position 113 is the last “S” of the C-terminal VTVSS motif,and positions 114, 115 and 116 are the positions immediately following(downstream) of said position 113.

To examine the effect of (other) substitutions in the C-terminal region,the influence of different substitutions was investigated by comparingrelated ISV's containing these substitutions, using the same analyticalpolyclonal antibody as described above. The analysis was done asdescribed above.

The ISVs containing said substitutions that were tested were NB's 3.1,3.2 and 3.4 (SEQ ID NO's 3, 4 and 5); NB's 3.10 to 3.15 (SEQ ID NO's 11to 16), which were compared with NB 3.4; NB's 4.1 and 4.2 (SEQ ID NO's17 and 18) and NB's 6.1, 6.2, 6.4 and 6.5 (SEQ ID NO's 19 to 22).

As representative examples of the kind of data obtained:

-   -   FIG. 4 shows the binding of NB's 3.4, 3.11, 3.12 and 3.13 to the        immobilized polyclonal antibody;    -   FIG. 5 shows the binding of NB's 3.4, 3.14 and 3.15 to the        immobilized polyclonal antibody;    -   FIG. 6 shows the binding of NB's 3.1, 3.2 and 3.4 to the        immobilized polyclonal antibody;    -   FIG. 7 shows the binding of NB's 4.1 and 4.2 to the immobilized        polyclonal antibody;    -   FIG. 8 shows the binding of NB's 6.1, 6.2, 6.4 and 6.5 to the        immobilized polyclonal antibody.

Tables VI, VII and VIII summarize the results obtained.

TABLE VI SEQ ID Position Position Position Binding** Clone ID NO 14⁽¹⁾83⁽¹⁾ 108⁽¹⁾ (RU) NB 3.4 5 P R L 75 NB 3.10 11 A R L 91 NB 3.11 12 P K L88 NB 3.12 13 A R Q 86 NB 3.13 14 P R Q 90 **Binding signal obtained atthe end of injection (=maximal RU signal) ⁽¹⁾numbering according toKabat.

TABLE VII SEQ ID Position Position Binding** Clone ID NO 11⁽¹⁾ 110⁽¹⁾(RU) NB 3.4 5 L T 75 NB 3.14 15 L Q 79 NB 3.15 16 S T 22 **Bindingsignal obtained at the end of injection (=maximal RU signal)⁽¹⁾numbering according to Kabat.

TABLE VIII SEQ ID Position Position Position Binding** Clone ID NO: 1483⁽¹⁾ 108⁽²⁾ Tag* (RU) NB 3.1 3 A K Q − 2 NB 3.2 4 A K Q + 0 NB 3.4 5 PR L − 59 Position Position Position Binding** Clone ID 14 83⁽³⁾ 108⁽⁴⁾Tag* (RU) NB 4.1 17 P R L − 51 NB 4.2 18 P R L + 0 Position PositionPosition Binding** Clone ID 14 83⁽⁵⁾ 108⁽⁶⁾ Tag* (RU) NB 6.1 19 P K Q +0 NB 6.2 20 P K Q − 39 NB 6.4 21 P R L + 0 NB 6.5 22 P R L − 66 *if “+”,this ISV contains additional amino acids at the C-terminal VTVSS end**Binding signal obtained at the end of injection (=maximal RU signal)⁽¹⁾numbering acc. to Kabat (corresponds to the a.a. at position 87 inSEQ ID NO's 3 to 5). ⁽²⁾numbering acc. to Kabat (corresponds to the a.a.at position 123 in SEQ ID NO's 3 to 5). ⁽³⁾numbering acc. to Kabat(corresponds to the a.a. at position 86 in SEQ ID NO's 17 and 18).⁽⁴⁾numbering acc. to Kabat (corresponds to the a.a. at position 116 inSEQ ID NO's 17 and 18). ⁽⁵⁾numbering acc. to Kabat (corresponds to thea.a. at position 86 in SEQ ID NO's 19 to 22). ⁽⁶⁾numbering acc. to Kabat(corresponds to the a.a. at position 112 in SEQ ID NO's 19 to 22).

Again, without being limited to any specific hypothesis or explanation,the data presented above shows that (various) substitutions to theC-terminal region (as defined herein) of an ISV can alter/improve itstendency to give rise to protein interference.

Example 4: Representative Protocols for Performing the ADA Assays ofFIG. 1

This Example gives some representative but non-limiting conditions thatcould be used for performing the competitive/bridging ADA assaysschematically shown in FIG. 1:

-   -   ADA assay of FIG. 1A in solution: Samples 100% matrix, 30′, 37°        C., Acid treatment using acetic acid in 10 matrix, 5′, RT,        Preincubation/acid neutralisation sample:ISV-Sulfo(:Tris) 1:1:1        (1:0.9:0.9:0.1), 1 h, RT; On plate 1 h, RT; Wash 3×, Readbuffer        4×    -   ADA assay of FIG. 1B in solution: Samples 20% matrix, 30′, 37°        C., Preincubation sample: ISV--Sulfo 1:1:1, 1 h, RT, On plate 1        h, RT, Wash 3×, Readbuffer 2×    -   Sequential ADA assay of FIG. 1C: Capture ISV-Bio, 1 h, RT, Wash        3×, Samples 20% matrix, 15′, RT, On plate: 2h, RT, Wash 3×,        Detection ALX-0141-Sulfo, 1 h, RT, Wash 3×, Readbuffer 4×

Example 5: Predicting Sensitivity of the ISV to Aspecific ProteinInterference Using the Analytical Antibody

This example describes a bridging/competition ADA assay using theanalytical antibody that can be used to predict sensitivity of an ISV toaspecific protein interference.

The ISV to be tested is diluted at a concentration of 10 μg/ml andincubated with the analytical antibody at 400 ng/ml, purified accordingto Example 2, and incubated at 37° C. at 600 rpm in 96 wellpolypropylene plates. The sample (50 μL) is then diluted ⅓ in 1:1mixture (100 μL) of 2 μg/ml biotinylated and 2 μg/ml sulfo-tagged ISVand incubated for 1 hour at RT, 600 RPM. MSD MA®96-well StandardStreptavidin plates are blocked with 150 μL/well Superblock® T20 for 1hour at RT, then washed 3 times with PBS/0.05% Tween20 (=wash buffer).Sample/1:1 mix (biotinylated and sulfo-tagged ISV) (50.0 μL) istransferred from the polypropylene plate to the MSD plate and incubatedfor 1 hour at RT, 600 rpm. Plates are washed three times prior toaddition of 2×Read Buffer (MSD) (150 μL/well) and reading the ECL units(ECLU) on an MSD instrument (Sector Imager 2400 reader).

Using this assay, the ISVs of SEQ ID NO's 23 to 30 were tested andcompared. The data are shown in Table IX. These data not only show thatthe assay described in this Example can be used to predict the tendencyof an ISV to give rise to protein interference, but the data generatedalso confirm the findings from the earlier Examples on the effect ofsubstitutions in the C-terminal region. As can be seen, addition of 3(and to lesser extent 1) Alanine residues at the C-terminus of the fullyhumanized ISV abolished its capacity to compete with binding of theanalytical antibody. Mutating position 14 on the wild type ISV variantfrom Alanine to Proline clearly increased its capacity as competitor inthe assay, (=making the ISV variant more prone to aspecific proteininterference), whereas mutating position 83 and 108 did not clearlyinfluenced the sensitivity of the ISV to aspecific protein interference.

TABLE IX ID affinity purified antibody IHuP#002-001-ABL-08 ECLU inscreening assay (using SEQ ID NO: 1) 2919 Nanobody Variant (right handcolumn mentions the humanizing substitutions and ECLU C-terminaladditions made compared to the confirmatory % wildtype sequence of SEQID NO: 23) assay reduction SEQ ID NO: 23 Wildtype VHH 2706 7.3 SEQ IDNO: 24 Wildtype VHH + A14P 268 90.8 SEQ ID NO: 25 Wildtype VHH + K83R2460 15.71 SEQ ID NO: 26 Wildtype VHH + Q108L 2533 13.23 SEQ ID NO: 27Wildtype VHH + A14P + 319 89.1 K83R + Q108L SEQ ID NO: 28 Wildtype VHH+A14P + 251 91.4 R39Q + K83R + T91Y + Q108L SEQ ID NO: 29 Wildtype VHH +A14P + 1207 58.64 R39Q + K83R + T91Y + Q108L + 1 additional A atC-terminus (A114) SEQ ID NO: 30 Wildtype VHH + A14P + 3301 −13.09 R39Q +K83R + T91Y + Q108L + 3 A's at C-terminus (A114 + A115 + A116)

Example 6: Influence of the Addition of Amino Acids to the C-Terminus ofAnti-OX40L Nanobodies on their OX40L Blocking Potency

This example demonstrates that the C-terminal extension has no influenceon activity or blocking potency of the Nanobodies.

The in vitro potency of the trivalent bispecific sequence optimizedanti-OX40L Nanobody Nb 3.16 (SEQ ID NO: 31) was compared with thepotency of the corresponding Nanobody containing one additional Ala atits C-terminus Nb 3.17 (SEQ ID NO: 32).

A first assay, the T-cell activation assay, was performed as follows.PBMCs were isolated from buffy coats (Red Cross, Ghent, Belgium) fromhealthy donors using Ficoll Paque Plus reagent (GE Healthcare) andwashed using RPMI 1640 complete medium (RPMI1640+GlutaMAX+25 mMHEPES+10% fetal bovine serum+1% Penicillin/Streptomycin; Invitrogen).The PBMC's (1×10⁵ cells/well) were stimulated with phytohaemagglutinin(PHA-L; final concentration 0.6 μg/ml) before the addition to 1×10⁴hOX40L expressing CHO cells (irradiated with gamma scintillator at 3000RAD; UZ Gent, Belgium) and dilution series of anti-OX40L Nanobodies RPMI1640 complete medium and incubated for 22 hours at 37° C. in CO₂incubator. Production of IL2 by the PBMCs was measured in ELISA. Wellsof a Maxisorp plate were coated overnight at 4° C. with anti-human IL2monoclonal antibody (BD Biosciences). After washing and blocking of thecoated wells, a ½ dilution of cell supernatant was added. As a standard,½ serial dilutions of recombinant human IL2 (BD Biosciences) startingfrom 2000 pg/ml were included. Detection was done using biotinylatedanti-human IL2 monoclonal antibody (BD Biosciences) and HRP conjugatedstreptavidin (Thermo Scientific) and esTMB (SDT Reagents). The reactionwas stopped with 1N HCl and the OD was read at 450 nm. As expected, thepotency of the trivalent bispecific sequence optimized Nanobody Nb 3.17(IC50=0.13 nM, 95% CI=0.098-0.17 nM) was comparable to that of Nb 3.16(IC50=0.10 nM, 95% CI=0.071-0.15 nM).

In a second ELISA-based competition assay, a dilution series (from 1.5μM to 0.083 pM) of the Nanobodies were pre-incubated overnight at roomtemperature with 100 ng/ml human OX40/Fc (R&D Systems) and 10 ng/mlbiotinylated human OX40L (R&D Systems; in-house biotinylated asdescribed in Example 1) in PBS+0.1% BSA+0.01% Tween-20. Next, thesamples were incubated on Maxisorp plates coated with 10 ug/mlanti-human Fc Nanobody (in-house generated) and blocked with PBS+1%BSA+0.1% Tween-20. Bound human OX40/Fc was detected using HRP conjugatedstreptavidin (Thermo Scientific) and sTMB (SDT Reagents). The reactionwas stopped with 1N HCl and the OD was read at 450 nm. In accordancewith the cell-based assay, the potency of the trivalent bispecificsequence optimized Nanobodies Nb 3.17 (IC50=0.178 nM, 95% CI=0.152-0.200nM) was comparable to that of Nb 3.16 (IC50=0.179 nM, 95% CI=0.149-0.215nM).

Example 7: Generation of Monoclonal Antibody 21-4-3

Two groups of different mice strains (BALB/c and NMRI—three mice each)were intraperitoneally immunized with the Nanobody construct of SEQ IDNO:98 in WO 2006/122825, in a water-in-oil emulsion of equal volumes ofantigen and Freund's complete or incomplete adjuvant) over a period of39 days, with boosting until suitable antiserum titers were obtained.

After asphyxiation of the stimulated mice in CO2, the spleens wereaseptically removed and a single cell suspension of pooled spleens wasprepared. Spleen cells and myeloma cells were washed several times withDMEM and fused in the presence of 1 ml 50% (w/v) PEG 3350 (ratio spleencells to SP2/0 3:1). For fusion was used the myeloma cell lineSP2/0-Ag14 from German Collection of Microorganisms and Cell Cultures(DSMZ GmbH, Braunschweig). This cell line is a hybrid between BALB/cspleen cells and the myeloma cell line P3x63Ag8. The so producedhybridomas were resuspended in CGM containing 20% FCS and aminopterin(HAT medium) and plated out (140 μl/well) into eight 96-well tissueculture flat-bottom plates (Corning-Costar) containing 140 μl/well CGM(20% FCS) with peritoneal excudate cells as feeder cells. The plateswere incubated for 10 days in a complete growth medium (CGM) containingDMEM with supplements 2-mercaptoethanol, L-Glutamin, Stable Glutamin, HTand non essential amino acids (in concentrations recommended by thesupplier) and FCS at different concentrations (10%, 15% or 20%). Duringthis period cells were fed two times with HAT medium. The cell culturesupernatants from hybridoma cells usually contained 1 to 20 μm/mlantibody, which were tested in a binding ELISA to confirm binding to theNanobody construct of SEQ ID NO:98 in WO 2006/122825.

Cells from positive IgG producing wells were transferred into wells of48 well plates and cultivated for 2-4 days (depending on growthcharacteristic of cells). Binding ELISA's on ALX081 and human/cynomolgusIgG were carried out in order to exclude the unspecific binders.Hybridoma cells expressing binders specific for the Nanobody constructof SEQ ID NO:98 in WO 2006/122825 were twice cloned using limiteddilution. After fusion and rescreening 7 primary cultures producingantibodies against ALX-081 were identified. All these primary culturesproduced antibodies not cross-reacting with human or cynomolgus IgG. Theprimary cultures were recloned (twice).

Clone 21-4 (one of the clones that stably produced antibodies againstALX-081 after the second cloning) was given the designation “ABH0015”and was deposited with the Belgian Coordinated Collections ofMicro-organisms (BCCM) in Ghent, Belgium on Jun. 4, 2012 under accessionnumber LMBP-9680-CB. The mouse monoclonal produced by ABH0015 was called21-4-3: isotype determination for 21-4-3 showed an IgG1 heavy chain anda kappa light chain, which were sequenced (see SEQ ID NO's: 35 and 36,respectively). 21-4-3 was shown to bind to the C-terminal region of theNanobody construct of SEQ ID NO:98 in WO 2006/122825 (data not shown).

Example 8: Binding of 21-4 to an ISV is Predictive of the Tendency of anISV to Undergo Aspecific Protein Interference

This Example together with the following Example 9 demonstrates thatbinding of the monoclonal 21-4 to an ISV can be used to predict (withinthe degrees of certainty indicated in this Example) of whether a givenISV will have a tendency to undergo aspecific protein interference (e.g.in an ADA assay).

This Example 8 in particular shows that 21-4 can be used to predictwhether certain proposed modifications to a given ISV (such as addingone or more amino acid residues to the C-terminus of an ISV and/orsubstituting one or more amino acid substitutions within the C-terminalregion of an ISV) will lead to a reduction of the tendency of said ISVto undergo aspecific protein interference.

In short, a set of 53 different Nanobodies and Nanobody constructs (seeFIG. 9 and SEQ ID NO's: 38 to 89) were tested for binding by monoclonal21-4-3. The same Nanobodies and Nanobody constructs were also tested forbinding by purified preparations of interference factor(s) obtained fromthree different human donors (referred to herein as “Donor 8”, “Donor19” and “Donor 30”), to see if there was any correlation between bindingby 21-4 and by the purified interference factors.

It was established that binding of an ISV by 21-4 can indeed be used topredict binding of the same ISV's by the interference factor(s) (withinthe overall degree of confidence provided by the data set out herein).

To demonstrate this, as detailed by the experimental data set out below,the binding of the 53 Nanobodies or Nanobody constructs (as listed inFIG. 9; see SEQ ID NO's: 38 to 89) by 21-4 was measured using a BiacoreT100 (according to the protocol set out below) and was compared tobinding of a reference Nanobody or construct (also listed in FIG. 9), asmeasured using the same Biacore instrument and the same protocol. Theresults are shown in Table X below.

TABLE X More than 70% More than 90% reduction of reduction of reductionof reduction in reduction in reduction in interference in interferencein interference in binding of binding of binding serum from serum fromserum from Nanobody to Nanobody to of 21-4-3 Donor A Donor B Donor C21-4-3 predicts 21-4-3 predicts vs binding compared to compared tocompared to >50% reduction >50% reduction SEQ C-terminal mutations to ofReference Reference Reference Reference in binding of in binding of IDamino the C-terminal Sequence Sequence Sequence Sequence nanobody tonanobody to NO: acid(s) region (=100%) (=100%) (=100%) (=100%)interference interference 37 A none  7%  3%  21%  31% ok ok 38 A none 0%  9%  25%  7% ok ok 39 A none  0%  10%  43%  35% ok ok 40 A none  1% 6% #N/A #N/A ok ok 41 A none  7%  6%  9% #N/A ok ok 42 A none  0%  1% 4% #N/A ok ok 43 A none  3%  3%  20% #N/A ok ok 44 A none  1%  5% #N/A#N/A ok ok 45 none P14A, P41T,  22%  0% #N/A #N/A ok S62F, S74A, S82bN,R83K, L108Q 46 AAEQKLI A14P, T41P,  2%  0% #N/A #N/A ok ok SEEDLN F62S,A74S, GAAHHH N82bS, K83R, HHH Q108L 47 GGGGSG none  4%  1% #N/A #N/A okok GGSRDW DFDVFG GGTPVG G 48 AAEQKLI none  3%  0% #N/A #N/A ok ok SEEDLNGAAHHH HHH 49 AAEQKLI V5L, I23A,  4%  0% #N/A #N/A OK OK SEEDLN E44G,A49S, GAAHHH A68T, A74S, HHH T78L, W79Y, K83R, T110Q, Q108L 50 none L11S 44%  77%  33% #N/A (<70% (<90% reduction) reduction) 51 none T110Q  88% 85%  84% #N/A (<70% (<90% reduction) reduction) 52 none S112G 100%  84% 58% #N/A (<70% (<90% reduction) reduction) 53 none S113G  13%  85%  88%#N/A NOK (<90% reduction) 54 none L11S, T110Q,  16%  39%  16% #N/A OK OKS112G, S113G 55 A none  6%  2%  21%  31% OK OK 56 G S113G  3%  2%  25% 0% OK OK 57 AS none  6%  1%  2% #N/A OK OK 58 AST none  6%  2%  2% #N/AOK OK 59 ASTK none  6%  2%  1% #N/A OK OK 60 ASP none  6%  2%  1% #N/AOK OK 61 AP none  6%  2%  2% #N/A OK OK 62 APT none  6%  2%  1% #N/A OKOK 63 W none  3%  4%  8% #N/A OK OK 64 L none  6%  3%  4% #N/A OK OK 65none P14A  23%  73% 121%  64% NOK (<90% reduction) 66 none L11S  48% 81%  29%  84% (<70% (<90% reduction) reduction) 67 none R83K 101% 102%117%  96% (<70% (<90% reduction) reduction) 68 none P14A, L108Q  26% 38% 115%  49% NOK (<90% reduction) 69 none L108Q 106%  80% 120%  84%(<70% (<90% reduction) reduction) 70 none T110Q 106%  90% 105%  98%(<70% (<90% reduction) reduction) 71 none S113G  44%  88% 105%  87%(<70% (<90% reduction) reduction) 72 none S112G, S113G  45%  70%  47% 56% (<70% (<90% reduction) reduction) 73 G S112G, S113G  1%  6%  8%  9%OK OK 74 G none  1%  4%  41%  33% OK OK 75 AA none  1%  1%  11%  16% OKOK 76 GGG none  2%  2%  17%  20% OK OK 77 A none  1%  2%  12%  13% OK OK78 none Q13R  1%  96%  96% 100% NOK NOK 79 GG none  2%  1%  17%  13% OKOK 80 none T110Q, S112G,  51%  65%  5%  58% (<70% (<90% S113G reduction)reduction) 81 none L11V  75%  94%  50%  89% (<70% (<90% reduction)reduction) 82 none P84A  56%  96%  80% 100% (<70% (<90% reduction)reduction) 83 none T87A  79%  56%  73%  61% (<70% (<90% reduction)reduction) 84 none S112G  91%  84%  50%  83% (<70% (<90% reduction)reduction) 85 none L11S, T110Q,  32%  46%  5%  42% (<70% (<90% S112G,S113G reduction) reduction) 86 none L11S, T110Q  64%  76%  6%  86% (<70%(<90% reduction) reduction) 87 none L11S, S112G,  41%  51%  5%  39%(<70% (<90% S113G reduction) reduction) 88 A L11S, T110Q  1%  1%  5% 11% OK OK 89 none L11S, P14A,  2%  14%  5%  17% OK OK T110Q, S112G,S113G

For each of the 53 Nanobodies or Nanobody constructs tested, thereference was chosen such that compared to the reference, the testedNanobodies or Nanobody constructs either had one or more additionalamino acid residues at the C-terminal end (which were added in order totest the effect of such addition on protein interference, and inparticular in order to reduce said interference) and/or one or moremutations within the C-terminal region (for example, as a result ofhumanization compared to the reference).

The results were expressed as a percentage reduction in binding(measured as RU units) for the given Nanobody versus the binding of thereference (also measured in RU units—for example, if the measuredbinding level (RU) of the reference Nanobody was 276 and the bindinglevel of the given Nanobody (also in RU) was 9, then the reduction inbinding level was to a level of [9 RU/276 RU]×100%=3%), which means areduction of 97% compared to the reference (100%).

Similarly, binding of the purified interference factor(s) from each ofthe three donors to each of the 53 Nanobodies or Nanobody constructs wasmeasured using the same Biacore instrument and compared to binding ofthe purified interference factor(s) to the same reference Nanobody orconstruct. The results were similarly expressed as a percentagereduction in binding of the interference factor to the given Nanobody orNanobody construct vs the reference.

It was found that for essentially all Nanobody or Nanobody construct inwhich one or more amino acid residues had been added to the C-terminalend compared to the reference, that the binding of the interferencefactor(s) was dramatically reduced. This again confirms that adding oneor more amino acid residues to the C-terminal end of an ISV (VTVSS) canreduce aspecific protein interference in an ADA assay. It was also foundthat in the majority of cases, only making substitutions within theC-terminal region (i.e. without adding one or more amino acid residuesto the C-terminus) compared to the reference often did not have asimilar dramatic impact on the binding of the interference factor(s).

The data was then further analysed to determine whether a reduction inbinding by 21-4 compared to the reference was in any way correlated witha reduction in binding by each of the three different preparations ofpurified interference factor compared to the reference. Suchcorrelations were found.

For example, it was found that of the 54 Nanobodies or Nanobodyconstructs tested, 36 showed a reduction in binding by 21-4 of more than70% compared to their respective reference sequence (with most of these36 having one or more additional amino acid residues at the C-terminus,in some cases in combination with substitutions within the C-terminalregion). Of these 36, 32 also showed reduction in binding by theinterference factor(s) compared to the reference of more than 50% (andin a large number of cases, in particular for Nanobodies or Nanobodyconstructs with one or more amino acid residues added at the C-terminus,the reduction was far greater than 50%, such as more than 70% or evenmore than 90%, see the data given in the Table X). This demonstratesthat in 32 out of 36 cases (i.e. 89%), a reduction in binding by 21-4 ofmore than 70% (compared to the reference=100%) is predictive for areduction in binding by the interference factors of more than 50%(compared to the same reference). For clarity, in each case, thereduction was calculated as 100%−[the percentage given in the Tablesbelow for the level of reduction achieved with the Nanobody tested].

Similarly, it was found that of the 53 Nanobodies or Nanobody constructstested, 33 showed a reduction in binding by 21-4 of more than 90%compared to their respective reference sequence (again, with most ofthese 33 having one or more additional amino acid residues at theC-terminus, in some cases in combination with substitutions within theC-terminal region). Of these 33, 32 also showed reduction in binding bythe interference factor(s) compared to their respective referencesequence of more than 50%. This demonstrates that in 32 out of 33 cases(i.e. 97%), a reduction in binding by 21-4 of more than 90% (compared tothe reference) is predictive for a reduction in binding by theinterference factors of more than 50% (compared to the same reference).

It should also be noted that such a reduction in binding of theinterference factor(s) by more than 50% (as evidenced by a reduction ofbinding by 21-4 of more than 70%) means that such interference factor(s)essentially no longer interfere(s) with an ADA assay for the ISV inquestion: experimental confirmation using an ADA assay showed that whenthe binding by the interference factor(s) is reduced by more than 45%,that no significant influence of the presence of the interferencefactor(s) on the ADA assay could be observed. In this respect, it willbe also be clear to the skilled person that this will even more so bethe case when the binding by interference factor(s) is reduced to anextent far greater than 50% (such as by more than 70% or even more than90%), as is observed in some cases (see again the data presentedherein).

In fact, it has been found that a reduction of more than 45% of bindingby 21-4 is indicative of a reduction of binding by interference factorsof more than 45%, which as mentioned means that the interferencefactor(s) no longer interfere with the ADA assay.

Moreover, the data presented herein on the correlation between(reduction in) binding by 21-4 and (reduction in) binding byinterference factor also allowed the present inventors to set anabsolute value for the binding by 21-4 below which it can be expected(within the confidence provided by the data set out in this Example 8)that an ISV or ISV-based construct will not be susceptible to binding byinterference factor(s) in a way that could interfere with an ADA assay.As set out in the following Example 9, this value is 500 RU (determinedand calculated as set out in Example 9).

Monoclonal 21-4 was purified from the culture medium of the hybridomaobtained in Example 7 above, as follows: Hybridoma cells secreting themonoclonal antibody 21-4-3 were cultured in spinner flasks in serum freemedium (CD Hybridoma, Gibco, supplemented with 8 mM L-glutamine(Invitrogen) and 1×cholesterol (250×cholersterol lipid concentrate,Gibco)) at a volume of 100 mL or 500 mL. The cleared supernatant wasfiltered, and the murine IgG1 captured on a ProteinA column (HiTrapMabSelect SuRe, 5 mL, GE Healthcare) at a reduced flow rate of 2 mL/min.Bound antibody was eluted in 0.1M citrate buffer pH3.0, and elutionfractions (of 5 mL) directly neutralized with 1 mL of 1M TRIS pH9.Purity of the antibody was verified by reducing and non-reducingSDS-PAGE.

The purified preparations of interference factor(s) from Donors 8 and 19were obtained from serum samples from said donors by means of affinitypurification, essentially as described in Example 2A. The interferencefactor(s) from Donor 30 were obtained from a serum sample of Donor 30,essentially as described in Example 2B.

To determine the binding of 21-4 to each of the Nanobodies or Nanobodyconstructs, the protocol described in Example 9 was used.

The binding of the interference factors from the three donors to each ofthe Nanobodies or Nanobody constructs was determined using a BiacoreT100 essentially as described in Example 3, using the interferencefactor from each of the donors 8, 19 and 30, directly immobilized on aCMS sensor chip.

Example 9: Protocol for Predicting Whether an ISV Will have a Tendencyto Undergo Aspecific Protein Interference (Using Monoclonal 21-4)

Binding measurements were performed using a Biacore T100 using a CM5T120416 sensor chip, with running buffer HBS-EP+, 25° C. 21-4 wascaptured via immobilized rabbit anti-mouse IgG, as it was found thatdirectly immobilized mAb 21-4-3 surface could not efficiently beregenerated. The anti-mouse IgG used was a polyclonal rabbit anti-mouseIgG antibodies reacting with all IgG subclasses, IgA and IgM (GEHealthcare; Cat #BR-1008-38; Lot #10056316). Immobilisation of theanti-mouse IgG was performed using manual amine coupling using a 7minute injection of EDC/NHS for activation and a 7 minute injection of1M ethanolamine HCl pH 8.5 for deactivation (Biacore, amine couplingkit). Binding conditions are listed in Table XI. Based on theimmobilization level and MW of the proteins, the theoretical R_(max) formAb21-4-3 binding to the immobilized anti-mouse IgG was ˜13000RU (whenone mAb21-4-3 molecule is binding to one anti-mouse IgG molecule).

TABLE XI Conc. Flow (μg/ Contact rate (μl/ Immobilization ImmobilizationProtein ml) time (s) min) buffer level (RU) Anti-mouse 30 420 5 10 mMacetate 13028 IgG pH 5.0 Anti-mouse 30 420 5 10 mM acetate 13318 IgG 24pH 5.0

The conditions used for the binding experiment (Biacore T100) using 21-4immobilized in the manner are given in Table XII. The anti-mouse IgGsurface could successfully be regenerated after capture of mAb21-4-3 andinjection of all samples (with a limited increase for baseline levelafter each regeneration).

TABLE XII Capture Flow path 4 Flow rate (μl/min) 10 Contact time (s) 180Concentration (μg/m1) 10 Binding and dissociation Flow path 3.4 Flowrate (μl/min) 45 Sample contact time (s) 120 Sample concentration (nM)500 Dissociation time (s) 600 Regeneration1 Flow path 3.4 Flow rate(μl/min) 10 Regeneration contact 180 time (s) Regeneration buffer 10 mMGlycine-HCl pH 1.7 Stabilization time (s) 120 If . . . Then . . . ElseIf after regeneration1 >20RU on Fc4 Else exit cycle Regeneration2 Flowpath 3.4 Flow rate (μl/min) 10 Regeneration contact 120 time (s)Regeneration buffer 10 mM Glycine-HCl pH 1.7 Stabilization time (s) 120

The above protocol was used to generate the 21-4 binding data set out inTable X. When the absolute values for RU were considered (afteradjusting the measured RU value for the molecular weight of the ISV,protein or polypeptide according to the formula ([RU measured]/[MW ofthe protein]×10⁶), it was found that the Nanobodies and Nanobodyconstructs mentioned in Table X that had an added alanine residue andthat showed >90% reduction in binding to both 21-4 as well asinterference factors, generally provided RU values of between 30RU and400RU (with the corresponding reference Nanobodies or polypeptides—aslisted in FIG. 9—having RU values of more than 1000, usually more than1500, and often more than 2000).

Based on this, it was considered that an (adjusted) RU value of lessthan 500 in this assay would be clearly indicative of an ISV (or aprotein or polypeptide that comprises as least one IS, as describedherein) that will (essentially) not be bound by interference factors ina manner that would interfere with an ADA assay.

The entire contents of all of the references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference, in particular for theteaching that is referenced herein.

The invention claimed is:
 1. A method of modifying a protein comprisingan amino acid sequence of an immunoglobulin single variable domain (VHor VHH) and ending with the sequence VTVSS (SEQ ID NO: 33) to havereduced aspecific protein binding to the C-terminal end of said protein,the method comprising extending the C-terminal end of said protein by 1to 5 amino acid residues to generate an extended protein ending with thesequence VTVSS(X)_(n)(SEQ ID NO: 34), in which n is 1 to 5, with each Xbeing independently chosen from any amino acid.
 2. The method of claim1, wherein the extended protein has reduced aspecific protein binding toits C-terminal end in a biological fluid of a human subject.
 3. Themethod of claim 1, wherein the biological fluid is a whole blood sample,a serum sample, a plasma sample, an ocular fluid sample, abronchoalveolar fluid sample, or a cerebrospinal fluid sample.
 4. Themethod of claim 1, wherein n is 1 to 5 , with each X being independentlychosen from any naturally occurring amino acid.
 5. The method of claim1, wherein n is 1 to 5 , with each X being independently chosen from thegroup consisting of alanine (A), glycine (G), valine (V), leucine (L)and isoleucine (I).
 6. The method of claim 5, wherein the amino acidsequence of the immunoglobulin single variable domain has valine atposition 11 according to Kabat numbering.
 7. The method of claim 1,wherein n is 1 or
 2. 8. The method of claim 1, in which: (a) n=1, 2 or 3in which each X=Ala or Gly; or (b) n=1, 2 or 3 in which each X=Ala; or(c) n=1, 2 or 3 in which each X=Gly; or (d) n=2 or 3 in which at leastone X=Ala or Gly, with any remaining amino acid residue X beingindependently chosen from any naturally occurring amino acid; or (e) n=2 or 3 in which all but one X=Ala or Gly, with any remaining amino acidresidue X being independently chosen from any naturally occurring aminoacid.
 9. The method of claim 8, in which in (d) or (e) the remainingamino acid residue X is independently chosen from the group consistingof alanine (A), glycine (G), valine (V), leucine (L) and isoleucine (I).10. The method of claim 8, wherein n=1 or n=2.
 11. The method of claim1, wherein the immunoglobulin single variable domain is a VHH orhumanized VHH.
 12. The method of claim 1, wherein the immunoglobulinsingle variable domain is a VH or a camelized VH.
 13. A method ofmodifying a protein comprising an amino acid sequence of animmunoglobulin single variable domain (VH or VHH) and ending with thesequence VTVSS (SEQ ID NO: 33) to have reduced aspecific protein bindingto the C-terminal end of said protein, the method comprising extendingthe C-terminal end of said protein by 1 to 5 amino acid residues togenerate an extended protein ending with the sequence VTVSS(X)_(n) (SEQID NO: 34), in which n is 1 to 5, with each X being independently chosenfrom any amino acid; wherein the protein comprises an amino acidsequence of two or more immunoglobulin single variable domains (VH orVHH), wherein at least one of the immunoglobulin single variable domainsbinds a therapeutic target.
 14. The method of claim 13, wherein theprotein further comprises an amino acid sequence of one or more linkersseparating the amino acid sequences of two or more immunoglobulin singlevariable domains of the protein.
 15. The method of claim 13, in which:(a) n=1, 2 or 3 in which each X=Ala or Gly; or (b) n=1, 2 or 3 in whicheach X=Ala; or (c) n=1, 2 or 3 in which each X=Gly; or (d) n=2 or 3 inwhich at least one X=Ala or Gly, with any remaining amino acid residue Xbeing independently chosen from any naturally occurring amino acid; or(e) n=2 or 3 in which all but one X=Ala or Gly, with any remaining aminoacid residue X being independently chosen from any naturally occurringamino acid.
 16. A method of modifying a protein comprising an amino acidsequence of an immunoglobulin single variable domain (VH or VHH) andending with the sequence VTVSS (SEQ ID NO: 33) to have reduced aspecificprotein binding to the C-terminal end of said protein, the methodcomprising extending the C-terminal end of said protein by 1 to 5 aminoacid residues to generate an extended protein ending with the sequenceVTVSS(X)_(n) (SEQ ID NO: 34), in which n is 1 to 5, with each X beingindependently chosen from any amino acid; wherein the protein comprisesan amino acid sequence of two or more immunoglobulin single variabledomains (VH or VHH), wherein one of the immunoglobulin single variabledomains binds serum albumin.
 17. The method of claim 16, wherein theimmunoglobulin single variable domain that binds serum albumin is at theC-terminal end of the protein.
 18. The method of claim 16, wherein theimmunoglobulin single variable domain that binds serum albumin is not atthe C-terminal end of the protein.
 19. The method of claim 16, wherein,the protein further comprises an amino acid sequence of one or morelinkers separating the amino acid sequences of two or moreimmunoglobulin single variable domains of the protein.
 20. The method ofclaim 16, in which: (a) n=1, 2 or 3 in which each X=Ala or Gly; or (b)n=1, 2 or 3 in which each X=Ala; or (c) n=1, 2 or 3 in which each X=Gly;or (d) n=2 or 3 in which at least one X=Ala or Gly, with any remainingamino acid residue X being independently chosen from any naturallyoccurring amino acid; or (e) n=2 or 3 in which all but one X=Ala or Gly,with any remaining amino acid residue X being independently chosen fromany naturally occurring amino acid.